Modulation of TIM receptor activity in combination with cytoreductive therapy

ABSTRACT

A genetic locus and corresponding family of proteins associated with regulation of immune function and cell survival are provided. These genes encode cell surface molecules with conserved IgV and mucin domains. The locus comprising the TIM family is genetically associated with immune dysfunction, including asthma. Furthermore, the TIM gene family is located within a region of human chromosome 5 that is commonly deleted in malignancies and myelodysplastic syndrome. Polymorphisms in the gene sequences are associated with the development of airway hyperreactivity and allergic inflammation, and T cell production of IL-4 and IL-13. The proteins include the human hepatitis A cellular receptor, hHAVcr-1.

BACKGROUND OF THE INVENTION

The T cell/transmembrane, immunoglobulin, and mucin (TIM) gene familywas positionally cloned in 2001 using a congenic mouse model of asthma.Since that time, a great deal of evidence has accumulated indicatingthat this gene family plays a critical role in regulating immuneresponses, including transplant tolerance, autoimmunity, the regulationof allergy and asthma, and the response to viral infections.

The TIM gene family consists of eight members (TIM-1-8) on mousechromosome 11B1.1, and three members (TIM-1, TIM-3, and TIM-4) on humanchromosome 5q33.2, located in a chromosomal region that has beenrepeatedly linked with asthma, allergy, and autoimmunity. Expression,function, and structural studies confirm that mouse TIM-1, TIM-3, andTIM-4 are the orthologues of human TIM-1, TIM-3, and TIM-4,respectively. TIM genes encode type I cell-surface glycoproteins withcommon structural features including an N-terminal immunoglobulin(Ig)-like domain, a mucin domain with O-linked glycosylations and withN-linked glycosylations close to the membrane, a single transmembranedomain, and a cytoplasmic region with tyrosine phosphorylation motif(s),except in TIM-4.

TIM-1, TIM-3, and TIM-4 are pattern recognition receptors specializedfor recognition of phosphatidylserine (PtdSer). PtdSer is normallylocalized to the inner leaflet of the plasma membrane but isredistributed and exposed on the outer membrane when a cell undergoesapoptosis. PtdSer on apoptotic cells provides a key signal that triggerscell engulfment. Recognition of apoptotic cells is an essentialcomponent of tissue homeostasis and immune regulation.

TIM-1, TIM-3, and TIM-4 differ in molecular structure and expressionpatterns, suggesting that they have distinct functions in regulatingT-cell responses. TIM-1, an important susceptibility gene for asthma andallergy, is preferentially expressed on Th2 cells and functions as apotent costimulatory molecule for T-cell activation. TIM-3 ispreferentially expressed on T-helper 1 (Th1) and Tc1 cells and generatesan inhibitory signal resulting in apoptosis of Th1 and Tc1 cells.Polymorphisms in TIM-1 and TIM-3 may reciprocally regulate the directionof T-cell responses. TIM-3 is also expressed on some dendritic cells(DCs) and can mediate phagocytosis of apoptotic cells andcross-presentation of antigen. In contrast, TIM-4 is exclusivelyexpressed on antigen-presenting cells (APCs), where it mediatesphagocytosis of apoptotic cells and plays an important role inmaintaining tolerance. TIM molecules thus provide a functionalrepertoire for recognition of apoptotic cells, which determines whetherapoptotic cell recognition leads to immune activation or tolerance,depending on the TIM molecule engaged and the cell type on which it isexpressed.

The development of therapies relating to this gene family is of greatclinical interest. The present invention addresses this issue.

RELATED PUBLICATIONS

The genetic sequence of the human hepatitis virus A cellular receptormay be found in Genbank, accession number XM_(—)011327. A relatedsequence is provided in Genbank, accession number BAB55044. Monney etal. (2002) Nature 415:436 describe cell surface molecules expressed onTh1 cells. U.S. Pat. Nos. 5,721,351, U.S. Pat. No. 6,204,371, U.S. Pat.No. 6,288,218 relate to sequences corresponding to a mouse TIM-3 allele.

SUMMARY OF THE INVENTION

Compositions and methods are provided for the treatment of cancer. Inthe methods of the invention, administration of a cytoreductive agent ortherapy, which include without limitation radiation therapy, e.g. localtumor radiation therapy; chemotherapy; and the like; in combination withadministration of an effective dose of an agent that is an modulator ofone or more TIM receptors, e.g. TIM-1, TIM-3 and TIM-4. In someembodiments the TIM modulator is a TIM-1 agonist, including withoutlimitation, activating antibodies. In some embodiments the TIM modulatoris a TIM-3 antagonist, including without limitation inhibitoryantibodies. In some embodiments the TIM modulator is a TIM-4 agonist,including without limitation, activating antibodies.

The dose of TIM modulator may be sufficient to enhance theanti-proliferative effects of the cytoreductive therapy on the targetedtumor, for example where the reduction in viable tumor cells followingtreatment is greater than the reduction in the absence of the TIMmodulator. The combination of TIM modulator and cytoreductive therapymay be synergistic, where the effectiveness of the combination isgreater than the additive activity of the TIM modulator or thecytoreductive therapy administered as a single modality.

The dose of TIM modulator may be sufficient to reduce the side effectsof the cytoreductive therapy on the patient, for example where there isa reduction in radiation-induced pneumonitis, hepatitis, and otherradiation-specific effects following treatment, relative to theside-effects in the absence of the TIM modulator.

In some embodiment of the invention, the administration of a TIMmodulator is guided by imaging with a phosphatidylserine (PS)-bindingagent, e.g. annexin V peptides, TIM fusion proteins or fragments, etc.,where the PS binding agent may be labeled for imaging, e.g. PET, SPECT,fluorescence, etc. The binding agent is brought into contact with thetarget tumor cells, where the presence of bound agent is indicative ofPS being present, and thus is indicative of the potential to activateTIM receptors on immune cells.

Genetic sequences of a gene family encoding polypeptides associated withimmune function and cell survival are provided. These genes encode cellsurface molecules with conserved IgV and mucin domains, herein referredto as T cell Immunoglobulin domain and Mucin domain (TIM) proteins. Thelocus comprising the TIM family is genetically associated with immunedysfunction, including asthma. Furthermore, the TIM gene family islocated within a region of human chromosome 5 that is commonly deletedin malignancies and myelodysplastic syndrome. Polymorphisms areidentified in TIM-1, TIM-3 and TIM-4, which can be associated withTh1/Th2 differentiation and airway hyperresponsiveness (AHR).

The nucleic acid compositions are used to produce the encoded proteins,which may be employed for functional studies, as a therapeutic, and instudying associated physiological pathways. TIM specific binding agents,including nucleic acids, antibodies, and the like, are useful asdiagnostics for determining genetic susceptibility to atopy and asthmaand as diagnostics for assessing tumor resistance to cancer therapy. TIMblocking agents find use as therapeutics in the treatment of immunedysfunction and disorders of cell survival, including malignancies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a, HBA mice produce significantly less IL-4 than do BALB/c mice.Lymph node cells of mice immunized with 150 μg of KLH were harvested,B-cell depleted, and cultured in cDME with 10 μg/ml KLH. Supernatantswere harvested after 96 hours and assessed for IL-4 levels by ELISA.Shown is a box plot of IL-4 levels (n=10 in each group), representingthe full range of data, with the boxes encompassing the upper and lowerquartiles, with the median of the data set shown inside each box. IL-4levels produced by BALB/c cells and by (BALB/c×HBA) F1 cells aresignificantly higher than HBA IL-4 levels P<0.0001 (student's t-test).b, HBA mice produce significantly less IL-13 and IL-10 than do BALB/cmice. Data shown are the average values of cytokines produced by lymphnode cell cultures from ten individual mice in each experimental group,±S.D. HBA IL-13 and IL-10 levels were lower than either the BALB/c or(BALB/c×HBA) F1 values, P<0.0001. HBA IL-5 versus BALB/c, P<0.05. HBAIFN-γ versus BALB/c, P<0.001. c, Allergen-induced airway hyperreactivityis significantly greater in BALB/c than do HBA mice. Pulmonary airflowobstruction was measured, and data shown represent peak enhanced pause(Penh) values averaged among sensitized mice in each group at variousmethacholine concentrations, ±S.E.M. (BALB×HBA) F1 demonstrate BALB/cphenotypes, while (BALB×DBA) F1 mice demonstrate DBA/2-like phenotypes.

FIG. 2. Regions of HBA chromosome 11 were inherited from DBA/2. HBAchromosome 11 contains two regions derived from DBA between hba-α2 andes-3, as delineated by SSLP markers. The regions of HBA chromosome 11with DBA/2 genotypes are highlighted (in blue) in the diagram to theleft. The markers (left) provide 2-5 cM resolution distal to D11Mit230.Where the mouse chromosome 11 has regions of highly conserved syntenywith 5q23-35, additional markers were identified with informativepolymorphisms between BALB/c and DBA/2, to provide 0.01-2 cM resolutionin this region (right column markers). Single-stranded confirmationpolymorphisms (SSCP) markers are shown in green to distinguish them fromthe SSLP markers, and the positions of particular genes of interest,denoted in red, are also shown. Where the arrangement of our marker mapdiffers from the Chromosome Committee Reports and the MIT linkage map,our map concurs with previous linkage and physical maps.

FIG. 3 a. IL-4 production by N2 mice is bimodal, with peakscorresponding to F1 and HBA phenotypes. As a means of evaluating therelative phenotypes of recombinant N2 mice in multiple experiments, weutilized an indexing function that allowed us to consolidate data frommultiple experiments. A histogram shows a bimodal distribution of IL-4index values for N2 mice with (BALB×HBA) F1, HBA homozygous, andrecombinant haplotypes indicated. Distributions associated with the F1and HBA haplotypes are distinct (P<0.0001, paired Student's t-test). b.IL-4 regulation segregates with Kim1sscp. Recombinant N2 haplotypes weresorted by IL-4 index values into groups associated with high IL-4phenotypes (index <0.35) and low IL-4 phenotypes (index >0.65). Eachcolumn of boxes represents a recombinant haplotype. Alleles for thesehaplotypes at loci between D11Mit269 to D11Mit154 are shaded accordingto genotype (dark=F1; pale=HBA). High IL-4 production segregates withfour haplotypes (left), and low IL-4 production segregates with fourhaplotypes (right). The IL-4 phenotype is linked to Kim1sscp. c. AHR ofN2 mice is bimodal, with peaks corresponding to F1 and HBA phenotypes.Index values calculated from Penh values are shown. The histogram showsa bimodal distribution of AHR index values for N2 mice. (BALB×HBA) F1,HBA homozygous, and recombinant haplotypes are indicated. Distributionsassociated with the F1 and HBA haplotypes are distinct (P<0.0001, pairedstudent's t-test). d. AHR Regulatory Locus cosegregates with the IL-4Regulatory Locus between D11Mit22 and D11Mit271. Recombinant N2haplotypes were sorted by AHR index values into groups associated withhigh AHR phenotypes (index <0.35) and low AHR phenotypes (index >0.65).Each column of boxes represents a recombinant haplotype. Alleles forthese haplotypes at loci between D11 Mit269 to D11Mit154 are shadedaccording to genotype (dark=F1; pale=HBA). High AHR segregates with fourhaplotypes (left), and low IL-4 production segregates with threehaplotypes (right). The AHR regulatory locus is linked to Kim1sscp,between D11Mit271 and D11Mit22.

FIG. 4 Mouse chromosome 11 interval containing Tapr is highly homologousto 5q33. In order to construct a composite map around the Tapr locus, weintegrated available information from the mouse linkage, backcross, andradiation hybrid maps and identified a region of highly conservedsynteny in current maps of the human genome (Human Genome Browser v3,UCSC, March 2001). ESTs located on a physical map of mouse chromosome 11(left) are denoted by their accession numbers and aligned by homology togenes (center), which correspond to ESTs on human chromosome 5 (right).All known genes between KIAA0171 and Sgd are shown.

FIG. 5 a,b,c. Identification novel TIM gene family and majorpolymorphisms in TIM-1 and TIM-3. FIG. 5 a depicts the amino acidsequences of mouse TIM-1 (SEQ ID NO:1); rat KIM-1 (SEQ ID NO:54); humanHAVcr-1 (SEQ ID NO:56) and monkey HAVcr-1 (SEQ ID NO: 55. FIG. 5Bdepicts an alignment of the amino acid sequences of mouse TIM-1 (SEQ IDNO:1), mouse TIM-2 (SEQ ID NO:5), and mouse a2-11 (SEQ ID NO:9). FIG. 5Cdepicts an alignment of TIM-1 variants, HBA (SEQ ID NO:3) and BALB/c(SEQ ID NO:1); and A2-11/TIM-3 variants, HBA (SEQ ID NO:11) and BALB/c(SEQ ID NO:9). Cloning of mouse TIM-1 and mouse TIM-2. Members of a genefamily. Sequences of the mouse TIM gene family members are shown. Shadedboxes illustrate identity between two of the mouse TIM genes. Total RNAfrom conA-stimulated splenocytes was reverse transcribed using GibcoSuperscript II. PCR products of full length Tim-3 cDNA were amplified,purified with Qiagen QIAquick PCR Purification reagents, and sequenceddirectly by Biotech Core (Mountain View, Calif.). PCR products for Tim-1and Tim-2 cDNA were cloned into electrocompetent TOP10 cells withTOPO-XL cloning reagents (Invitrogen). Plasmids were purified with astandard alkaline lysis protocol. BALB/c and HBA plasmids weresequenced, as described. Homology of mouse TIM-1, rat KIM-1, andHAVcr-1. Identity with mouse TIM-1 is denoted by the shaded boxes. Theapproximate signal site is denoted by an open, inverted triangle and theIg domain/mucin domain boundary is shown with a filled diamond. Thepredicted transmembrane domains are underlined. TIM-1 and TIM-3sequences with major polymorphisms between BALB/c and HBA TIM-1 andTIM-3 shown.

FIG. 6. Tapr Regulates CD4 T cell IL-4 and IL-13 Responses. T cells fromBALB/c DO11.10 mice produce higher levels of IL-4 and IL-13 in responseto antigen than do T cells from HBA DO11.10 mice. Splenic CD4+ cellswere isolated by positive selection with anti-CD4 magnetic beads andthen cocultured with bone marrow-derived DCs and OVA. After seven days,the cells were restimulated. Supernatants were harvested after 18-24hours of the secondary culture. Data represent mean cytokine levelsdetected at increasing concentrations of OVA, ±S.D. Detection ofexpression of TIM-1 mRNA in purified CD 4 T cells during priming anddifferentiation.

FIG. 7. Polymorphisms in human TIM-1, NM 012206 (SEQ ID NO:17).

FIG. 8. SSCP polymorphism analysis of human TIM-1.

FIG. 9. A schematic for assessing the effect of combining radiation andimmune therapies.

FIG. 10. Graph of survival probability.

FIG. 11. Day 55 liver histology demonstrates that the agonisticanti-TIM1 antibody 3B3 depletes distant metastatic 4T1 breast cancercells and associated immunosuppressive MSC inflammation better thananti-CTLA4.

FIG. 12. Side effect of radiation therapy in the absence and presence ofimmunotherapy.

DETAILED DESCRIPTION OF THE EMBODIMENTS

New, more effective, and less toxic therapies are needed to treatcancer, including carcinomas such as breast cancer, prostate cancer andthe like. The methods of the invention provide for a combination ofcytoreductive therapy, including radiation therapy such as local tumorradiation therapy, which serves to kill irradiated cancer cells andreleases antigens in close proximity to immune cells in tumors, withimmunotherapy (IT), which promotes a local immune response against theirradiated tumor and leads the immune system to respond to sites ofmetastatic disease outside of the irradiation field.

Radiation therapy is known to enhance antigen presentation and T cellresponses to antigen presenting cells. Factors controlling T cellactivation by APCs presenting tumor antigen include TCR:MHC interaction,costimulation, and cytokines. Costimulation is determined by acollection of costimulatory and coinhibitory receptor/ligand pairsresiding at the cell surfaces of T cells and antigen presenting cells.In order for an effective adaptive immune response to occur and togenerate immune memory, costimulation is required. CD28, ICOS, HVEM,CD27, CD30, CD40L, OX40, 4-1 BB, TIM-1, and SLAM are major costimulatoryreceptors.

The methods of the invention utilize TIM receptor modulation as animmune therapy (IT) that, when combined with radiation (RT) or othercytoreductive therapy to the primary tumor site, enhances local cancertumor control and reduces the size and number of distant cancermetastases. Since this approach uses RT to one site to kill disease atmultiple sites, and uses the immune system to kill cancer cellsthroughout the body, it can reduce the need for RT to multiple sites ofdisease and additional chemotherapy.

Unlike locally administered adjuvants such as CpG oligonucleotides (CpGODN), cytokines, or dendritic cells which typically require direct(invasive) tumoral manipulation, costimulation-enhancing monoclonalantibodies can be administered as a single dose intravenously and‘boost’ the local response after RT without an invasive procedure. Inthis manner, an IT would be used concurrently with local RT directed atan internal tumor target or a single symptomatic metastasis, promote aRT-associated immune response, and generate a systemic (abscopal) immuneresponse.

Because TIM receptors can be activated by binding to phosphatidylserine,a phospholipid that is exposed in irradiated tumor tissues, IT with TIMreceptors may demonstrate greater specificity (and fewer ‘off target’autoimmune side effects) than other IT agents currently underdevelopment, and the functional interaction between TIM-1 and PS in thetumor immune microenvironment provides a unique method for focusingimmune activation to the irradiated tumor.

The TIM-1 gene and protein have several known polymorphisms thatinfluence the function of the TIM-1 in immune responses. These geneticvariations are most common in the mucin domain that is involved withTIM-1 adhesion to extracellular molecules and plays an important role inT helper cell function. Mice with functional deletions in exon 4 ofTIM-1 develop less robust helper T cell responses. In human populationsa polymorphism involving exon 4 encodes a longer form of the mucinchain, and this polymorphism impacts immune function. This polymorphismin TIM-1 has been linked to susceptibility to asthma and autoimmunity.Polymorphisms determining the length of the mucin domain may be analyzedprior to treatment involving stimulation of a TIM receptor.

Tumors of interest for treatment with the methods of the inventioninclude solid tumors, e.g. carcinomas, gliomas, melanomas, sarcomas, andthe like. Breast cancer is of particular interest. Carcinomas includethe a variety of adenocarcinomas, for example in prostate, lung, etc.;adernocartical carcinoma; hepatocellular carcinoma; renal cellcarcinoma, ovarian carcinoma, carcinoma in situ, ductal carcinoma,carcinoma of the breast, basal cell carcinoma; squamous cell carcinoma;transitional cell carcinoma; colon carcinoma; nasopharyngeal carcinoma;multilocular cystic renal cell carcinoma; oat cell carcinoma, large celllung carcinoma; small cell lung carcinoma; etc. Carcinomas may be foundin prostrate, pancreas, colon, brain (usually as secondary metastases),lung, breast, skin, etc. Including in the designation of soft tissuetumors are neoplasias derived from fibroblasts, myofibroblasts,histiocytes, vascular cells/endothelial cells and nerve sheath cells.Tumors of connective tissue include sarcomas; histiocytomas; fibromas;skeletal chondrosarcoma; extraskeletal myxoid chondrosarcoma; clear cellsarcoma; fibrosarcomas, etc.

Anti-proliferative, or cytoreductive therapy is used therapeutically toeliminate tumor cells and other undesirable cells in a host, andincludes the use of therapies such as delivery of ionizing radiation,and administration of chemotherapeutic agents. Chemotherapeutic agentsare well-known in the art and are used at conventional doses andregimens, or at reduced dosages or regimens, including for example,topoisomerase inhibitors such as anthracyclines, including the compoundsdaunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin,anamycin, MEN 10755, and the like. Other topoisomerase inhibitorsinclude the podophyllotoxin analogues etoposide and teniposide, and theanthracenediones, mitoxantrone and amsacrine. Other anti-proliferativeagent interferes with microtubule assembly, e.g. the family of vincaalkaloids. Examples of vinca alkaloids include vinblastine, vincristine;vinorelbine (NAVELBINE); vindesine; vindoline; vincamine; etc.DNA-damaging agent include nucleotide analogs, alkylating agents, etc.Alkylating agents include nitrogen mustards, e.g. mechlorethamine,cyclophosphamide, melphalan (L-sarcolysin), etc.; and nitrosoureas, e.g.carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU),streptozocin, chlorozotocin, etc. Nucleotide analogs includepyrimidines, e.g. cytarabine (CYTOSAR-U), cytosine arabinoside,fluorouracil (5-FU), floxuridine (FUdR), etc.; purines, e.g. thioguanine(6-thioguanine), mercaptopurine (6-MP), pentostatin, fluorouracil (5-FU)etc.; and folic acid analogs, e.g. methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, etc. Otherchemotherapeutic agents of interest include metal complexes, e.g.cisplatin (cis-DDP), carboplatin, oxaliplatin, etc.; ureas, e.g.hydroxyurea; and hydrazines, e.g. N-methylhydrazine.

For example, ionizing radiation (IR) is used to treat about 60% ofcancer patients, by depositing energy that injures or destroys cells inthe area being treated, and for the purposes of the present inventionmay be delivered at conventional doses and regimens, or at reduceddoses. Radiation injury to cells is nonspecific, with complex effects onDNA. The efficacy of therapy depends on cellular injury to cancer cellsbeing greater than to normal cells. Radiotherapy may be used to treatevery type of cancer. Some types of radiation therapy involve photons,such as X-rays or gamma rays. Another technique for delivering radiationto cancer cells is internal radiotherapy, which places radioactiveimplants directly in a tumor or body cavity so that the radiation doseis concentrated in a small area. A suitable dose of ionizing radiationmay range from at least about 2 Gy to not more than about 10 Gy, usuallyabout 5 Gy. A suitable dose of ultraviolet radiation may range from atleast about 5 J/m² to not more than about 50 J/m², usually about 10J/m². The sample may be collected from at least about 4 and not morethan about 72 hours following ultraviolet radiation, usually aroundabout 4 hours.

The TIM family genes are immediately adjacent to each other on humanchromosome 5, in the order TIM-4, TIM-1, TIM-3, with no interveninggenes. This segment of human chromosome 5 is commonly deleted inmalignancies and dysplastic cell populations, as in myeolodysplasticsyndrome (see Boultwood, et al, (1997) Genomics 45:88-96). There are TIMpseudogenes on chromosomes 5, 12, and 19. Each TIM protein, exceptTIM-4, contains a distinct predicted tyrosine signaling motif. Thecytoplasmic region of TIM-1 contains two tyrosine residues and includesa highly conserved tyrosine kinase phosphorylation motif, (SEQ ID NO:59)RAEDNIY. The expanded region, (SEQ ID NO:60) SRAEDNIYIVEDRP, contains apredicted site for Itk phosphorylation and for EGF-receptorphosphorylation. The activity of TIM polypeptides may be modulated inorder to direct immune function. TIM-1 is preferentially expressed inTh2 cells, and agents that modulate TIM-1 activity find use in thetreatment of Th2 related disorders, including allergies, asthma, and thelike. TIM-3 is preferentially expressed in Th1 cells, and agents thatmodulate TIM-3 activity find use in the treatment of pro-inflammatoryimmune diseases, including autoimmune diseases, graft rejection and thelike.

Agents that modulate activity of TIM genes or proteins provide a pointof therapeutic or prophylactic intervention, particularly agents thatinhibit or upregulate activity of the polypeptide, or expression of thegene, particularly in combination with cytoreductive therapy. Numerousagents are useful in modulating this activity, including agents thatdirectly modulate expression, e.g. expression vectors, antisensespecific for the targeted polypeptide; and agents that act on theprotein, e.g. specific antibodies and analogs thereof, small organicmolecules that block catalytic activity, etc. The use of antibodies orspecific binding fragments derived therefrom is of particular interest,e.g. antibodies specific for TIM-1, TIM-3 or TIM-4.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host tomodulate TIM function, e.g. to enhance TIM function. The therapeuticagents may be administered in a variety of ways, orally, topically,parenterally e.g. intravenous, subcutaneously, intraperitoneally, byviral infection, intravascularly, etc. Intravenous delivery is ofparticular interest. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways. The concentration oftherapeutically active compound in the formulation may vary from about0.1-100 wt. %.

In some embodiment of the invention, the administration of a TIMmodulator is guided by imaging with a phosphatidylserine (PS)-bindingagent, e.g. annexin V peptides, TIM fusion proteins or fragments, etc.,where the PS binding agent may be labeled for imaging, e.g. PET, SPECT,fluorescence, etc. The binding agent is brought into contact with thetarget tumor cells, where the presence of bound agent is indicative ofPS being present, and thus is indicative of the potential to activateTIM receptors on immune cells.

For example, Annexin V is a ubiquitous intracellular protein in humansthat has a nanomolar affinity for the membrane-bound constitutiveanionic phospholipid phosphatidylserine (PS), which is selectivelyexpressed on the surface of apoptotic or physiologically stressed cells.As such, radiolabeled forms of annexin V have been used in both animalmodels and human Phase I and Phase II trials for utilizing the tracer asan early surrogate marker of therapeutic efficacy. For example seeBlankenberg et al. (2009) Proc. Am. J. Thoracic. Soc. 6:469-476, hereinspecifically incorporated by reference. As the TIM proteins also bind tophosphatidylserine, the annexin V labeling is a useful surrogate, orother molecules that specifically bind to PS, e.g. TIM selectivemarkers.

For example, the in vivo monitoring PS exposure (ie TIM targetexpression) after radiation therapy may be performed with anti-annexin Vlinked to a label radiotracer (for PET or SPECT), or with a TIM fusionprotein linked to a label or radiotracer. Labels could include q-dotsfor near IR imaging, or other more conventional labels. pK/Kd studiesmay be performed with radiolabelled antibodies, using methods asdescribed by Blankenberg for annexin V imaging.

The TIM modulatory agent may be administered prior to, concurrentlywith, or following the cytoreductive therapy, usually within at leastabout 1 week, at least about 5 days, at least about 3 days, at leastabout 1 day. The TIM modulatory agent may be delivered in a single dose,or may be fractionated into multiple doses, e.g. delivered over a periodof time, including daily, bidaily, semi-weekly, weekly, etc. Theeffective dose will vary with the route of administration, the specificagent, the dose of cytoreductive agent, and the like, and may bedetermined empirically by one of skill in the art. A useful range fori.v. administered antibodies may be empirically determined, for exampleat least about 0.1 mg/kg body weight; at least about 0.5 mg/kg bodyweight; at least about 1 mg/kg body weight; at least about 2.5 mg/kgbody weight; at least about 5 mg/kg body weight; at least about 10 mg/kgbody weight; at least about 20 mg/kg body weight; or more.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

TIM Gene Family

The provided TIM family genes and fragments thereof, encoded proteins,genomic regulatory regions, and specific antibodies are useful in theidentification of individuals predisposed to development or resistanceto asthma, and for the modulation of gene activity in vivo forprophylactic and therapeutic purposes. The encoded proteins are usefulas an immunogen to raise specific antibodies, in drug screening forcompositions that mimic or modulate activity or expression, includingaltered forms of the proteins, and as a therapeutic.

The mouse Tim1 gene encodes a 305 amino acid membrane protein. Thecytoplasmic region of TIM-1 contains two tyrosine residues and includesa highly conserved tyrosine kinase phosphorylation motif, RAEDNIY. Themucin domain of TIM-1 has multiple sites for O-linked glycosylation, andthere two sites for N-linked glycosylation found in the immunoglobulindomain.

Mouse TIM-2, a similar 305 amino acid membrane protein, has 64% identityto mouse TIM-1, 60% identity to rat KIM-1, and 32% identity to hHAVcr-1.Like TIM-1, TIM-2 has two extracellular N-linked glycosylation sites anda serine, threonine-rich mucin domain with many O-linked glycosylationsites. TIM-2 also has an intracellular tyrosine kinase phosphorylationmotif, (SEQ ID N0:61) RTRCEDQVY.

Tim3 encodes a 281 amino acid membrane protein in mice, and a 301 aminoacid protein in humans, that has a similar, integral membraneglycoprotein structure with multiple extracellular glycosylation sitesand an intracellular tyrosine phosphorylation motif. Although the mucindomain is not as prominent in TIM-3 as it is in TIM-1 and TIM-2, TIM-3expressed on T cells interacts with a ligand on APCs and alters APCactivation. TIM-3 has four sites for N-linked and five sites forO-linked glycosylation, suggesting that TIM-3, like TIM-1 and TIM-2, isheavily glycosylated and might interact with a ligand present on othercells, such as antigen presenting cells.

Tim4 encodes a 344 amino acid protein in mice, and a 378 amino acidprotein in humans. The predicted TIM-4 also shares the general membraneglycoprotein structural motifs of the other TIM proteins, a with anIgV-like domain with highly conserved cysteine residues, athreonine-rich mucin-like domain, and a short intracellular tail.

Polymorphisms in the murine sequences are provided in the sequencelisting for the BALB/c and HBA/DBA strains. In TIM-1, thesepolymorphisms encode three amino acid differences and a fifteen aminoacid deletion in HBA/DBA. Seven predicted amino acid differences wereidentified in TIM-3. The polymorphisms in TIM-1 and TIM-4 are located inthe signal and mucin-like domains, while the polymorphisms identified inTIM-3 are clustered in the Ig domain.

Variants in coding regions of human Tim1 are provided in the seqlist andFIG. 8. Variations include an insertion (labeled polymorphism 1),157insMTTTVP, observed in 65% of the chromosomes, and a deletion(polymorphism 5), 187ΔThr, observed in 65% of the chromosomes. Otherpolymorphisms are T140A (polymorphism 7); V161A; (polymorphism 2); V167I(polymorphism 3); T172A (polymorphism 4); N258D (polypmorphism 6).Polymorphism 4 was observed in 40% of the chromosomes, and the otherpolymorphisms were each observed in ≦5% of the chromosomes. Most ofthese variations (2-6) are located within exon 3, the firstmucin-encoding exon, and all of the variants occur at the genomic leveland are not splice variants. The association between Tim1 and asthmasusceptibility is further supported by reports of significant linkage ofmite-sensitive childhood asthma to D5S820 (mean LOD score=4.8), a markerwhich is approximately 0.5 megabases from Tim1.

In human tissues, a 4.4 kb TIM-1 mRNA is present in almost all tissues,though it is faint in most. A 5.5-kb band was observed in colon andliver. A 7.5-kb band was observed in spleen, thymus, and peripheralblood leukocytes, and smaller than 4.4-kb bands were observed in someorgans. TIM-1 mRNA is expressed with alternate 5′ untranslated regions,in different cell populations. Hypoxia and ischemia induces TIM-1expression in epithelial cells, and radiation induces expression of TIMgene family mRNA. The TIM genes are expressed in tumor specimens. HumanTIM-4 mRNA is expressed in glioblastoma tissue, and is also detected inmitogen stimulated or irradiated peripheral blood monocytes.

In one aspect, the invention provides for an isolated nucleic acidmolecule other than a naturally occurring chromosome comprising asequence encoding a TIM-1, TIM-2, TIM-3 or TIM-4 protein, or a homologor variant thereof, which variant may be associated with susceptibilityto airway hyperreactivity and allergic T cell responses. The nucleicacid may be operably linked to a vector and/or control sequences forexpression in a homologous or heterologous host cell. Such a host cellcan find use in the production of the encoded protein. In another aspectof the invention, a purified polypeptide is provided of TIM-1, TIM-2,TIM-3 or TIM-4 protein, or a homolog or variant thereof, which variantmay be associated with susceptibility to airway hyperreactivity andallergic T cell responses. In another aspect, an antibody or otherspecific binding member that binds to the TIM-1, TIM-2, TIM-3 or TIM-4polypeptide is provided.

The DNA sequence encoding Tim polypeptides may be cDNA or genomic DNA ora fragment thereof. Fragments of interest for probes, producingpolypeptides, etc. may comprise one or more polymorphic residues. Theterm Tim gene shall be intended to mean the open reading frame encodingany one of the specific Tim polypeptides, introns, as well as adjacent5= and 3= non-coding nucleotide sequences involved in the regulation ofexpression, up to about 1 kb beyond the coding region, but possiblyfurther in either direction. The gene may be introduced into anappropriate vector for extrachromosomal maintenance or for integrationinto the host.

In some embodiments, the Tim gene sequence is other than human TIM-1allele 1, as set forth in the sequence listing; and/or other than mouseTIM-3 DBA allele.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 3= and 5= non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns removed by nuclear RNA splicing, to create acontinuous open reading frame encoding a Tim protein.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3= and 5=untranslated regions found in the mature mRNA. It may further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5= or 3= end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence.

The sequence of the 5′ region, and further 5′ upstream sequences and 3′downstream sequences, may be utilized for promoter elements, includingenhancer binding sites, that provide for expression in tissues where Timgenes are expressed. The tissue specific expression is useful fordetermining the pattern of expression, and for providing promoters thatmimic the native pattern of expression. Naturally occurringpolymorphisms in the promoter region are useful for determining naturalvariations in expression, particularly those that may be associated withdisease. Alternatively, mutations may be introduced into the promoterregion to determine the effect of altering expression in experimentallydefined systems. Methods for the identification of specific DNA motifsinvolved in the binding of transcriptional factors are known in the art,e.g. sequence similarity to known binding motifs, gel retardationstudies, etc. For examples, see Blackwell et al. (1995) Mol Med 1:194-205; Mortlock et al. (1996) Genome Res. 6: 327-33; and Joulin andRichard-Foy (1995) Eur J Biochem 232: 620-626.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of TIMexpression, especially in different tissues or stages of development,and to identify cis acting sequences and trans acting factors thatregulate or mediate TIM expression. Such transcription or translationalcontrol regions may be operably linked to a TIM gene in order to promoteexpression of wild type or altered TIM or other proteins of interest incultured cells, or in embryonic, fetal or adult tissues, and for genetherapy.

The nucleic acid compositions of the subject invention may encode all ora part of the subject polypeptides. Fragments may be obtained of the DNAsequence by chemically synthesizing oligonucleotides in accordance withconventional methods, by restriction enzyme digestion, by PCRamplification, etc. For the most part, DNA fragments will be of at least15 nt, usually at least 18 nt, more usually at least about 50 nt. Suchsmall DNA fragments are useful as primers for PCR, hybridizationscreening, etc. Larger DNA fragments, i.e. greater than 100 nt areuseful for production of the encoded polypeptide. For use inamplification reactions, such as PCR, a pair of primers will be used.The exact composition of the primer sequences is not critical to theinvention, but for most applications the primers will hybridize to thesubject sequence under stringent conditions, as known in the art. It ispreferable to choose a pair of primers that will generate anamplification product of at least about 50 nt, preferably at least about100 nt. Algorithms for the selection of primer sequences are generallyknown, and are available in commercial software packages. Amplificationprimers hybridize to complementary strands of DNA, and will primetowards each other.

The TIM genes are isolated and obtained in substantial purity, generallyas other than an intact mammalian chromosome. Usually, the DNA will beobtained substantially free of other nucleic acid sequences that do notinclude an TIM sequence or fragment thereof, generally being at leastabout 50%, usually at least about 90% pure and are typicallyArecombinant@, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

The DNA sequences are used in a variety of ways. They may be used asprobes for identifying TIM related genes. Mammalian homologs havesubstantial sequence similarity to the subject sequences, i.e. at least75%, usually at least 90%, more usually at least 95% sequence identitywith the nucleotide sequence of the subject DNA sequence. Sequencesimilarity is calculated based on a reference sequence, which may be asubset of a larger sequence, such as a conserved motif, coding region,flanking region, etc. A reference sequence will usually be at leastabout 18 nt long, more usually at least about 30 nt long, and may extendto the complete sequence that is being compared. Algorithms for sequenceanalysis are known in the art, such as BLAST, described in Altschul etal. (1990) J Mol Biol 215:403-10.

Nucleic acids having sequence similarity are detected by hybridizationunder low stringency conditions, for example, at 50° C. and 10×SSC (0.9M saline/0.09 M sodium citrate) and remain bound when subjected towashing at 55° C. in 1×SSC. Sequence identity may be determined byhybridization under stringent conditions, for example, at 50° C. orhigher and 0.1×SSC (9 mM saline/0.9 mM sodium citrate). By using probes,particularly labeled probes of DNA sequences, one can isolate homologousor related genes. The source of homologous genes may be any species,e.g. primate species, particularly human; rodents, such as rats andmice, canines, felines, bovines, ovines, equines, yeast, Drosophila,Caenhorabditis, etc.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature and does not require elaborationhere. mRNA is isolated from a cell sample. mRNA may be amplified byRT-PCR, using reverse transcriptase to form a complementary DNA strand,followed by polymerase chain reaction amplification using primersspecific for the subject DNA sequences. Alternatively, mRNA sample isseparated by gel electrophoresis, transferred to a suitable support,e.g. nitrocellulose, nylon, etc., and then probed with a fragment of thesubject DNA as a probe. Other techniques, such as oligonucleotideligation assays, in situ hybridizations, and hybridization to DNA probesarrayed on a solid chip may also find use. Detection of mRNA hybridizingto the subject sequence is indicative of TIM gene expression in thesample.

The subject nucleic acid sequences may be modified for a number ofpurposes, particularly where they will be used intracellularly, forexample, by being joined to a nucleic acid cleaving agent, e.g. achelated metal ion, such as iron or chromium for cleavage of the gene;or the like.

The sequence of the TIM locus, including flanking promoter regions andcoding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or product of such a mutation will besubstantially similar to the sequences provided herein, i.e. will differby at least one nucleotide or amino acid, respectively, and may differby at least two but not more than about ten nucleotides or amino acids.The sequence changes may be substitutions, insertions or deletions.Deletions may further include larger changes, such as deletions of adomain or exon. Other modifications of interest include epitope tagging,e.g. with the FLAG system, HA, etc. For studies of subcellularlocalization, fusion proteins with green fluorescent proteins (GFP) maybe used. Such mutated genes may be used to study structure-functionrelationships of TIM polypeptides, or to alter properties of the proteinthat affect its function or regulation. For example, constitutivelyactive transcription factors, or a dominant negatively active proteinthat binds to the TIM DNA target site without activating transcription,may be created in this manner.

Techniques for in vitro mutagenesis of cloned genes are known. Examplesof protocols for scanning mutations may be found in Gustin et al.,Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli etal., Mol Gen Genet. 199:537-9 (1985); and Prentki et al., Gene 29:303-13(1984). Methods for site specific mutagenesis can be found in Sambrooket al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.15.3-15.108; Weiner et al., Gene 126:35-41 (1993); Sayers et al.,Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55 (1990);Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu Anal Biochem177:120-4 (1989).

Arrays provide a high throughput technique that can assay a large numberof polynucleotides in a sample. In one aspect of the invention, an arrayis constructed comprising one or more of the TIM genes, proteins orantibodies, preferably comprising all of these sequences, which arraymay further comprise other sequences known to be up- or down-regulatedin T cells, monocytes, and the like. This technology can be used as atool to test for differential expression, or for genotyping. Arrays canbe created by spotting polynucleotide probes onto a substrate (e.g.,glass, nitrocellulose, etc.) in a two-dimensional matrix or array havingbound probes. The probes can be bound to the substrate by eithercovalent bonds or by non-specific interactions, such as hydrophobicinteractions. Techniques for constructing arrays and methods of usingthese arrays are described in, for example, Schena et al. (1996) ProcNatl Acad Sci USA. 93(20):10614-9; Schena et al. (1995) Science270(5235):467-70; Shalon et al. (1996) Genome Res. 6(7):639-45, U.S.Pat. No. 5,807,522, EP 799 897; WO 97/29212; WO 97/27317; EP 785 280; WO97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP 728 520;U.S. Pat. No. 5,599,695; EP 721 016; U.S. Pat. No. 5,556,752; WO95/22058; and U.S. Pat. No. 5,631,734.

The probes utilized in the arrays can be of varying types and caninclude, for example, synthesized probes of relatively short length(e.g., a 20-mer or a 25-mer), cDNA (full length or fragments of gene),amplified DNA, fragments of DNA (generated by restriction enzymes, forexample) and reverse transcribed DNA. Both custom and generic arrays canbe utilized in detecting differential expression levels. Custom arrayscan be prepared using probes that hybridize to particular preselectedsubsequences of mRNA gene sequences or amplification products preparedfrom them.

Arrays can be used to, for example, examine differential expression ofgenes and can be used to determine gene function. For example, arrayscan be used to detect differential expression, or expression ofpolymorphic sequences, in TIM genes. Exemplary uses of arrays arefurther described in, for example, Pappalarado et al. (1998) Sem.Radiation Oncol. 8:217; and Ramsay. (1998) Nature Biotechnol. 16:40.Furthermore, many variations on methods of detection using arrays arewell within the skill in the art and within the scope of the presentinvention. For example, rather than immobilizing the probe to a solidsupport, the test sample can be immobilized on a solid support which isthen contacted with the probe. Additional discussion regarding the useof microarrays in expression analysis can be found, for example, inDuggan, et al., Nature Genetics Supplement 21:10-14 (1999); Bowtell,Nature Genetics Supplement 21:25-32 (1999); Brown and Botstein, NatureGenetics Supplement 21:33-37 (1999); Cole et al., Nature GeneticsSupplement 21:38-41 (1999); Debouck and Goodfellow, Nature GeneticsSupplement 21:48-50 (1999); Bassett, Jr., et al., Nature GeneticsSupplement 21:51-55 (1999); and Chakravarti, Nature Genetics Supplement21:56-60 (1999).

Pharmacogenetics is the linkage between an individual's genotype andthat individual's ability to metabolize or react to a therapeutic agent.Differences in metabolism or target sensitivity can lead to severetoxicity or therapeutic failure by altering the relation betweenbioactive dose and blood concentration of the drug. In the past fewyears, numerous studies have established good relationships betweenpolymorphisms in metabolic enzymes or drug targets, and both responseand toxicity. These relationships can be used to individualizetherapeutic dose administration.

Genotyping of polymorphic alleles is used to evaluate whether anindividual will respond well to a particular therapeutic regimen. Thepolymorphic sequences are also used in drug screening assays, todetermine the dose and specificity of a candidate therapeutic agent. Acandidate TIM polymorphism is screened with a target therapy todetermine whether there is an influence on the effectiveness in treatingasthma. Drug screening assays are performed as described above.Typically two or more different sequence polymorphisms are tested forresponse to a therapy.

The subject gene may be employed for synthesis of a complete TIMprotein, or polypeptide fragments thereof, particularly fragmentscorresponding to functional domains; binding sites; etc.; and includingfusions of the subject polypeptides to other proteins or parts thereof.For expression, an expression cassette may be employed, providing for atranscriptional and translational initiation region, which may beinducible or constitutive, where the coding region is operably linkedunder the transcriptional control of the transcriptional initiationregion, and a transcriptional and translational termination region.Various transcriptional initiation regions may be employed that arefunctional in the expression host.

Polypeptides of particular interest that are fragments of the TIMpolypeptides include specific domains of the TIM polypeptides, where adomain may comprise, for example, the extracellular domain, or thedomains within the extracellular domain: the mucin domain and/or the Igdomain. Domains may also comprise the cytoplasmic domain, e.g. afragment encompassing the tyrosine kinase phosphorylation motif, (SEQ IDNO:59) RAEDNIY, or the expanded region, SEQ ID NO:60) SRAEDNIYIVEDRP.Polypeptides encoded by the soluble splice variants are also ofinterest. The sequence of the Ig domains are as follows: human TIM-1 Igdomain, SEQ ID NO: 17, 19, 21, 23, 25, 27, residues 21-126; human TIM-3Ig domain, SEQ ID NO: 29 and 31, residues 22-131; human TIM-4 Ig domain,SEQ ID NO: 33 and 35, residues 25-133; mouse TIM-1 Ig domain, SEQ ID NO:1 and 3, residues 21-129; mouse TIM-2 Ig domain, SEQ ID NO: 7, residues22-128; mouse TIM-3 Ig domain, BALB/c allele, SEQ ID NO: 9, residues22-132; mouse TIM-3 Ig domain, DBA/2 allele, SEQ ID No: 11, residues22-132; mouse TIM-4 Ig domain, SEQ ID NO: 13 and 15, residues 25-135.

Functionally equivalent polypeptides may find use, where the equivalentpolypeptide may contain deletions, additions or substitutions of aminoacid residues that result in a silent change, thus producing afunctionally equivalent differentially expressed on pathway geneproduct. Amino acid substitutions may be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity, and/orthe amphipathic nature of the residues involved. “Functionallyequivalent”, as used herein, refers to a protein capable of exhibiting asubstantially similar in vivo activity as the polypeptide encoded by aTIM gene.

The polypeptides may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, may be used as the expression host cells. In many situations, itmay be desirable to express the TIM gene in mammalian cells, where theTIM gene will benefit from native folding and post-translationalmodifications. Small peptides can also be synthesized in the laboratory,including specific peptide epitopes, domains, and the like, wherepeptides will usually be at least about 8 amino acids in length, moreusually at least about 20 amino acids in length, up to complete domains,and the full length protein. Peptides may comprise polymorphic regionsof the protein. Also included are fusion proteins, where all or afragment of the TIM protein is fused to a heterologous polypeptide, e.g.green fluorescent protein, antibody Fc regions, poly-histidine, and thelike.

In mammalian host cells, a number of viral-based expression systems maybe used, including retrovirus, lentivirus, adenovirus, adeno-associatedvirus, and the like. In cases where an adenovirus is used as anexpression vector, the coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing differentially expressed or pathway gene protein in infectedhosts.

Specific initiation signals may also be required for efficienttranslation of the genes. These signals include the ATG initiation codonand adjacent sequences. In cases where a complete gene, including itsown initiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of thegene coding sequence is inserted, exogenous translational controlsignals must be provided. These exogenous translational control signalsand initiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc.

With the availability of the polypeptides in large amounts, by employingan expression host, the polypeptides may be isolated and purified inaccordance with conventional ways. A lysate may be prepared of theexpression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. The purified polypeptide will generally be atleast about 80% pure, preferably at least about 90% pure, and may be upto and including 100% pure. Pure is intended to mean free of otherproteins, as well as cellular debris.

The polypeptide may be labeled, either directly or indirectly. Any of avariety of suitable labeling systems may be used, including but notlimited to, radioisotopes such as ¹²⁵I; enzyme labeling systems thatgenerate a detectable colorimetric signal or light when exposed tosubstrate; and fluorescent labels. Indirect labeling involves the use ofa protein, such as a labeled antibody, that specifically binds to thepolypeptide of interest. Such antibodies include but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression library.

Specific Binding Members

The term “specific binding member” or “binding member” as used hereinrefers to a member of a specific binding pair, i.e. two molecules,usually two different molecules, where one of the molecules (i.e., firstspecific binding member) through chemical or physical means specificallybinds to the other molecule (i.e., second specific binding member). Thecomplementary members of a specific binding pair are sometimes referredto as a ligand and receptor; or receptor and counter-receptor. For thepurposes of the present invention, the two binding members may be knownto associate with each other, for example where an assay is directed atdetecting compounds that interfere with the association of a knownbinding pair. Alternatively, candidate compounds suspected of being abinding partner to a compound of interest may be used.

Specific binding pairs of interest include carbohydrates and lectins;complementary nucleotide sequences; peptide ligands and receptor;effector and receptor molecules; hormones and hormone binding protein;enzyme cofactors and enzymes; enzyme inhibitors and enzymes; lipid andlipid-binding protein; etc. The specific binding pairs may includeanalogs, derivatives and fragments of the original specific bindingmember. For example, a receptor and ligand pair may include peptidefragments, chemically synthesized peptidomimetics, labeled protein,derivatized protein, etc.

In a preferred embodiment, the specific binding member is an antibody,which may activate the TIM receptor, as an agonist antibody, or mayinhibit the Tim receptor, as an inhibitor antibody. The term “antibody”or “antibody moiety” is intended to include any polypeptidechain-containing molecular structure with a specific shape that fits toand recognizes an epitope, where one or more non-covalent bindinginteractions stabilize the complex between the molecular structure andthe epitope. Antibodies that bind specifically to one of the TIMproteins are referred to as anti-TIM. The archetypal antibody moleculeis the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA,IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow,sheep, pig, dog, other mammal, chicken, other avians, etc., areconsidered to be “antibodies.” Antibodies utilized in the presentinvention may be polyclonal antibodies, although monoclonal antibodiesare preferred because they may be reproduced by cell culture orrecombinantly, and can be modified to reduce their antigenicity.

Polyclonal antibodies can be raised by a standard protocol by injectinga production animal with an antigenic composition, which may be apolypeptide or a cDNA expressed in vivo. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.When utilizing an entire protein, or a larger section of the protein,antibodies may be raised by immunizing the production animal with theprotein and a suitable adjuvant (e.g., Fruend's, Fruend's complete,oil-in-water emulsions, etc.) When a smaller peptide is utilized, it isadvantageous to conjugate the peptide with a larger molecule to make animmunostimulatory conjugate. Commonly utilized conjugate proteins thatare commercially available for such use include bovine serum albumin(BSA) and keyhole limpet hemocyanin (KLH). In order to raise antibodiesto particular epitopes, such as polymorphic residues, peptides derivedfrom the full sequence may be utilized. The immunogen is injected intothe animal host, preferably according to a predetermined scheduleincorporating one or more booster immunizations, and the animals arebled periodically. Polyclonal antibodies may then be purified from suchantisera by, for example, affinity chromatography using the polypeptidecoupled to a suitable solid support.

Alternatively, for monoclonal antibodies, hybridomas may be formed byisolating the stimulated immune cells, such as those from the spleen ofthe inoculated animal. These cells are then fused to immortalized cells,such as myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. The immortal cell line utilized ispreferably selected to be deficient in enzymes necessary for theutilization of certain nutrients. Many such cell lines (such asmyelomas) are known to those skilled in the art, and include, forexample: thymidine kinase (TK) or hypoxanthine-guanine phosphoriboxyltransferase (HGPRT). These deficiencies allow selection for fused cellsaccording to their ability to grow on, for example, hypoxanthineaminopterinthymidine medium (HAT).

In addition, the antibodies or antigen binding fragments may be producedby genetic engineering. In this technique, as with the standardhybridoma procedure, antibody-producing cells are sensitized to thedesired antigen or immunogen. The messenger RNA isolated from the immunespleen cells or hybridomas is used as a template to make cDNA using PCRamplification. A library of vectors, each containing one heavy chaingene and one light chain gene retaining the initial antigen specificity,is produced by insertion of appropriate sections of the amplifiedimmunoglobulin cDNA into the expression vectors. A combinatorial libraryis constructed by combining the heavy chain gene library with the lightchain gene library. This results in a library of clones which co-expressa heavy and light chain (resembling the Fab fragment or antigen bindingfragment of an antibody molecule). The vectors that carry these genesare co-transfected into a host (e.g. bacteria, insect cells, mammaliancells, or other suitable protein production host cell). When antibodygene synthesis is induced in the transfected host, the heavy and lightchain proteins self-assemble to produce active antibodies that can bedetected by screening with the antigen or immunogen.

Chimeric antibodies may be made by recombinant means by combining themurine variable light and heavy chain regions (VK and VH), obtained froma murine (or other animal-derived) hybridoma clone, with the humanconstant light and heavy chain regions, in order to produce an antibodywith predominantly human domains. The production of such chimericantibodies is well known in the art, and may be achieved by standardmeans (as described, e.g., in U.S. Pat. No. 5,624,659, incorporatedfully herein by reference). Humanized antibodies are engineered tocontain even more human-like immunoglobulin domains, and incorporateonly the complementarity-determining regions of the animal-derivedantibody. This is accomplished by carefully examining the sequence ofthe hyper-variable loops of the variable regions of the monoclonalantibody, and fitting them to the structure of the human antibodychains. Although facially complex, the process is straightforward inpractice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully hereinby reference.

Alternatively, polyclonal or monoclonal antibodies may be produced fromanimals that have been genetically altered to produce humanimmunoglobulins. Techniques for generating such animals, and derivingantibodies therefrom, are described in U.S. Pat. Nos. 6,162,963 and6,150,584, incorporated fully herein by reference.

Alternatively, single chain antibodies (Fv, as described below) can beproduced from phage libraries containing human variable regions. SeeU.S. Pat. No. 6,174,708. Intrathecal administration of single-chainimmunotoxin, LMB-7 [B3(Fv)-PE38], has been shown to cure ofcarcinomatous meningitis in a rat model. Proc Natl. Acad. Sci. USA 92,2765-9, all of which are incorporated by reference fully herein.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by pepsin, papain, or otherprotease cleavage. “Fragment,” or minimal immunoglobulins may bedesigned utilizing recombinant immunoglobulin techniques. For instance“Fv” immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

Fv fragments are heterodimers of the variable heavy chain domain (V_(H))and the variable light chain domain (V_(L)). The heterodimers of heavyand light chain domains that occur in whole IgG, for example, areconnected by a disulfide bond. Recombinant Fvs in which V_(H) and V_(L)are connected by a peptide linker are typically stable. These are singlechain Fvs which have been found to retain specificity and affinity andhave been shown to be useful for imaging tumors and to make recombinantimmunotoxins for tumor therapy. However, researchers have bound thatsome of the single chain Fvs have a reduced affinity for antigen and thepeptide linker can interfere with binding. Improved Fv's have been alsobeen made which comprise stabilizing disulfide bonds between the V_(H)and V_(L) regions, as described in U.S. Pat. No. 6,147,203, incorporatedfully herein by reference. Any of these minimal antibodies may beutilized in the present invention, and those which are humanized toavoid HAMA reactions are preferred for use in embodiments of theinvention.

In addition, derivatized immunoglobulins with added chemical linkers,detectable moieties, such as fluorescent dyes, enzymes, substrates,chemiluminescent moieties and the like, or specific binding moieties,such as streptavidin, avidin, or biotin, and the like may be utilized inthe methods and compositions of the present invention. For convenience,the term “antibody” or “antibody moiety” will be used throughout togenerally refer to molecules which specifically bind to an epitope ofthe brain tumor protein targets, although the term will encompass allimmunoglobulins, derivatives, fragments, recombinant or engineeredimmunoglobulins, and modified immunoglobulins, as described above.

Candidate antibodies can be tested for activity by any suitable standardmeans. As a first screen, the antibodies may be tested for bindingagainst the immunogen. As a second screen, antibodies may be screenedfor cross-reactivity between alleles and between TIM family members, andtested for activity in inhibition of TIM function. For these screens,the candidate antibody may be labeled for detection. Antibodies thatalter the biological activity of a TIM protein may be assayed infunctional formats.

Diagnosis

Diagnosis of conditions associated with Tim polymorphisms is performedby protein, DNA or RNA sequence and/or hybridization analysis of anyconvenient sample from a patient, e.g. biopsy material, blood sample,scrapings from cheek, etc. A nucleic acid sample from a patient having acondition that may be associated with TIM, is analyzed for the presenceof a polymorphism in TIM. A typical patient genotype would have at leastone mutation on at least one chromosome. Individuals are screened byanalyzing their DNA or mRNA for the presence of a predisposingpolymorphism, as compared to an asthma neutral sequence. Specificsequences of interest include any polymorphism that leads to clinicalbronchial hyperreactivity or is otherwise associated with asthma,including, but not limited to, insertions, substitutions and deletionsin the coding region sequence, intron sequences that affect splicing, orpromoter or enhancer sequences that affect the activity and expressionof the protein. Examples of specific TIM polymorphisms are provided inthe Examples.

Screening may also be based on the functional or antigeniccharacteristics of the protein. Immunoassays designed to detectpredisposing polymorphisms in TIM proteins may be used in screening.Where many diverse mutations lead to a particular disease phenotype,functional protein assays have proven to be effective screening tools.

Biochemical studies may be performed to determine whether a candidatesequence polymorphism in the TIM coding region or control regions isassociated with disease. For example, a change in the promoter orenhancer sequence that affects expression of TIM may result inpredisposition to asthma. Expression levels of a candidate variantallele are compared to expression levels of the normal allele by variousmethods known in the art. Methods for determining promoter or enhancerstrength include quantitation of the expressed natural protein;insertion of the variant control element into a vector with a reportergene such as βBgalactosidase, luciferase, chloramphenicolacetyltransferase, etc. that provides for convenient quantitation; andthe like. The activity of the encoded TIM protein may be determined bycomparison with the wild-type protein.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence. Where large amounts of DNA areavailable, genomic DNA is used directly. Alternatively, the region ofinterest is cloned into a suitable vector and grown in sufficientquantity for analysis. Cells that express TIM genes, such as tracheacells, may be used as a source of mRNA, which may be assayed directly orreverse transcribed into cDNA for analysis. The nucleic acid may beamplified by conventional techniques, such as the polymerase chainreaction (PCR), to provide sufficient amounts for analysis. The use ofthe polymerase chain reaction is described in Saiki, et al. (1985)Science 239:487, and a review of current techniques may be found inSambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 14.2B14.33. Amplification may also be used to determine whether apolymorphism is present, by using a primer that is specific for thepolymorphism. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. (1990) N.A.R. 18:2887-2890; and Delahuntyet al. (1996) Am. J. Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2=,7=-dimethoxy-4=,5=-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2=,4=,7=,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N═,N=-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzedby one of a number of methods known in the art. The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to a neutral TIM sequence. Hybridization with the variantsequence may also be used to determine its presence, by Southern blots,dot blots, etc. The hybridization pattern of a control and variantsequence to an array of oligonucleotide probes immobilised on a solidsupport, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, mayalso be used as a means of detecting the presence of variant sequences.Single strand conformational polymorphism (SSCP) analysis, denaturinggradient gel electrophoresis (DGGE), mismatch cleavage detection, andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease (restrictionfragment length polymorphism, RFLP), the sample is digested with thatendonuclease, and the products size fractionated to determine whetherthe fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels.

The hybridization pattern of a control and variant sequence to an arrayof oligonucleotide probes immobilised on a solid support, as describedin U.S. Pat. No. 5,445,934, or in WO95/35505, may be used as a means ofdetecting the presence of variant sequences. In one embodiment of theinvention, an array of oligonucleotides are provided, where discretepositions on the array are complementary to at least a portion of mRNAor genomic DNA of the TIM locus. Such an array may comprise a series ofoligonucleotides, each of which can specifically hybridize to a nucleicacid, e.g. mRNA, cDNA, genomic DNA, etc. from the TIM locus.

Antibodies specific for TIM polymorphisms may be used in screeningimmunoassays. A reduction or increase in neutral TIM and/or presence ofasthma associated polymorphisms is indicative that asthma isTIM-associated. A sample is taken from a patient suspected of havingTIM-associated asthma. Samples, as used herein, include biologicalfluids such as tracheal lavage, blood, cerebrospinal fluid, tears,saliva, lymph, dialysis fluid and the like; organ or tissue culturederived fluids; and fluids extracted from physiological tissues. Alsoincluded in the term are derivatives and fractions of such fluids.Biopsy samples are of particular interest, e.g. trachea scrapings, etc.The number of cells in a sample will generally be at least about 10³,usually at least 10⁴ more usually at least about 10⁵. The cells may bedissociated, in the case of solid tissues, or tissue sections may beanalyzed. Alternatively a lysate of the cells may be prepared.

Diagnosis may be performed by a number of methods. The different methodsall determine the absence or presence or altered amounts of normal orabnormal TIM in patient cells suspected of having a predisposingpolymorphism in TIM. For example, detection may utilize staining ofcells or histological sections, performed in accordance withconventional methods. The antibodies of interest are added to the cellsample, and incubated for a period of time sufficient to allow bindingto the epitope, usually at least about 10 minutes. The antibody may belabeled with radioisotopes, enzymes, fluorescers, chemiluminescers, orother labels for direct detection. Alternatively, a second stageantibody or reagent is used to amplify the signal. Such reagents arewell known in the art. For example, the primary antibody may beconjugated to biotin, with horseradish peroxidase-conjugated avidinadded as a second stage reagent. Final detection uses a substrate thatundergoes a color change in the presence of the peroxidase. The absenceor presence of antibody binding may be determined by various methods,including flow cytometry of dissociated cells, microscopy, radiography,scintillation counting, etc.

An alternative method for diagnosis depends on the in vitro detection ofbinding between antibodies and TIM in a lysate. Measuring theconcentration of TIM binding in a sample or fraction thereof may beaccomplished by a variety of specific assays. A conventional sandwichtype assay may be used. For example, a sandwich assay may first attachTIM-specific antibodies to an insoluble surface or support. Theparticular manner of binding is not crucial so long as it is compatiblewith the reagents and overall methods of the invention. They may bebound to the plates covalently or non-covalently, preferablynon-covalently.

Other immunoassays are known in the art and may find use as diagnostics.Ouchterlony plates provide a simple determination of antibody binding.Western blots may be performed on protein gels or protein spots onfilters, using a detection system specific for TIM as desired,conveniently using a labeling method as described for the sandwichassay.

The TIM genes are useful for analysis of TIM expression, e.g. indetermining developmental and tissue specific patterns of expression,and for modulating expression in vitro and in vivo. Vectors useful forintroduction of the gene include plasmids and viral vectors. Ofparticular interest are retroviral-based vectors, e.g. Moloney murineleukemia virus and modified human immunodeficiency virus; adenovirusvectors, etc. that are maintained transiently or stably in mammaliancells. A wide variety of vectors can be employed for transfection and/orintegration of the gene into the genome of the cells. Alternatively,micro-injection may be employed, fusion, or the like for introduction ofgenes into a suitable host cell. See, for example, Dhawan et al. (1991)Science 254:1509-1512 and Smith et al. (1990) Molecular and CellularBiology 3268-3271.

The expression vector will have a transcriptional initiation regionoriented to produce functional mRNA. The native transcriptionalinitiation region or an exogenous transcriptional initiation region maybe employed. The promoter may be introduced by recombinant methods invitro, or as the result of homologous integration of the sequence into achromosome. Many strong promoters are known in the art, including theβ-actin promoter, SV40 early and late promoters, human cytomegaloviruspromoter, retroviral LTRs, methallothionein responsive element (MRE),tetracycline-inducible promoter constructs, etc.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences. Transcription cassettes may be prepared comprising atranscription initiation region, the target gene or fragment thereof,and a transcriptional termination region. The transcription cassettesmay be introduced into a variety of vectors, e.g. plasmid; retrovirus,e.g. lentivirus; adenovirus; and the like, where the vectors are able totransiently or stably be maintained in the cells, usually for a periodof at least about one day, more usually for a period of at least aboutseveral days to several weeks.

Antisense molecules are used to down-regulate expression of TIM incells. The anti-sense reagent may be antisense oligonucleotides (ODN),particularly synthetic ODN having chemical modifications from nativenucleic acids, or nucleic acid constructs that express such anti-sensemolecules as RNA. The antisense sequence is complementary to the mRNA ofthe targeted gene, and inhibits expression of the targeted geneproducts. Antisense molecules inhibit gene expression through variousmechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996) Nature Biotechnology 14:840-844).

Compound Screening

One can identify ligands or substrates that bind to, modulate or mimicthe action of TIM. Areas of investigation are the development oftreatments for immune disorders, asthma, cancer, ischemia-reperfusioninjury, and other diseases that are associated with cellular responsesto stress. Drug screening identifies agents that provide an inhibition,replacement, or enhancement for TIM function in affected cells. Forexample, agents that reverse or inhibit TIM function may reducebronchial reactivity in asthma by reducing levels of Th2 cytokines, andTIM inhibitors may enhance tumor sensitivity to cancer therapy, bypotentiating the effects of radiation and chemotherapeutic treatmentsthat induce apoptosis. Of particular interest are screening assays foragents that have a low toxicity for human cells. A wide variety ofassays may be used for this purpose, including labeled in vitroprotein-protein binding assays, protein-DNA binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,and the like. The purified protein may also be used for determination ofthree-dimensional crystal structure, which can be used for modelingintermolecular interactions, transcriptional regulation, etc.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of altering or mimicking thephysiological function of TIM, such as a signal tyrosine kinaseinhibitor, or a peptide inhibitor of an integrin binding site. Generallya plurality of assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e. at zero concentration or below the level ofdetection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including, but not limited to: peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

Other assays of interest detect agents that mimic TIM function. Forexample, candidate agents are added to a cell that lacks functional TIM,and screened for the ability to reproduce TIM in a functional assay.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the array” includes reference to one or more arrays andequivalents thereof known to those skilled in the art, and so forth. Alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the celllines, constructs, and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXPERIMENTAL

To analyze the human 5q23-35 region for asthma susceptibility genes, weutilized a mouse model, which offers several potential advantages.Environmental variation can be controlled, multiple phenotypes can betested simultaneously, and inbred strains can be sensitized withallergen to develop airway hyperreactivity (AHR), a cardinal feature ofhuman asthma. We utilized congenic inbred mouse strains that differedonly by a small chromosomal region homologous to human chromosome 5q,thereby allowing this region to be studied in the absence of geneticvariation outside the region. Positional cloning revealed a novel genefamily encoding T cell membrane proteins (Tim), TIM-1, TIM-2, TIM-3,TIM-4, TIM-5, TIM-6, and TIM-7, in which major sequence variants ofTIM-1, TIM-3, and TIM-4, cosegregate completely with Tapr.

IL-4 production and airway hyperreactivity are reduced in HBA mice. Weexamined congenic mice produced on a BALB/c genomic background withdiscrete genomic intervals inherited from individual DBA/2 chromosomes.BALB/c mice develop Th2 biased, atopy-resembling immune responses withenhanced AHR, while DBA/2 mice develop reduced IL-4 responses thatprotect against the development of AHR. By screening several of thesecongenic strains for reduced Th2 responsiveness, we identified onecongenic line, C.D2 Es-Hba (HBA), which contained a segment ofchromosome 11 inherited from DBA/2 mice, homologous to human 5q23-35.FIG. 1 a shows that lymph node cells from immunized control BALB/c mice,as expected, produced high levels of IL-4, confirming the proclivity ofBALB/c mice to develop Th2-biased immune responses. In contrast, lymphnode cells from HBA mice produced significantly lower levels of IL-4,similar to that observed in DBA/2 mice. In addition, HBA mice producedsignificantly less IL-13 and IL-10, and somewhat lower levels of IL-5compared to BALB/c mice, whereas production of IFN-γ was increased, asshown in FIG. 1 b. These results indicated that the DBA/2-derived regionof HBA chromosome 11, which has large regions of conserved synteny withhuman 5q23-35, contains a gene that reduces antigen-specific IL-4,IL-13, and IL-10 production, enhances IFN-γ production, and converts theBALB/c cytokine phenotype into a DBA/2 cytokine phenotype.

The HBA mice were examined for the capacity to develop antigen-inducedairway hyperreactivity (AHR), which is associated with Th2-biased immuneresponses. Upon sensitization and challenge with allergen, controlBALB/c mice developed high AHR, whereas similarly immunized HBA congenicmice, like DBA/2 mice, expressed normal airway reactivity in response tomethacholine (FIG. 1 c). Collectively, these results strongly suggestedthat genetic variation in a single locus on chromosome 11 regulated bothTh2 cytokine production and AHR; therefore, we tentatively refer to therelevant genetic determinant(s) in HBA mice as a single locus, T celland Airway Phenotype Regulator (Tapr).

We also examined (BALB/c×HBA) F1 mice, which like the BALB/c mice,produced high levels of IL-4, IL-13, and IL-10 (FIGS. 1 a and 1 b) anddeveloped elevated antigen-induced AHR (FIG. 1 c). These resultsindicate that a DBA/2 allele on chromosome 11, in isolation of othergenes that regulate IL-4 synthesis, reduced IL-4 production and AHR in arecessive manner. In contrast, (BALB/c×DBA/2) F1 mice produced lowlevels of IL-4 and had normal airway responsiveness on immunization(FIG. 1), indicating that loci from other regions of the DBA genome alsomodulated IL-4 production and antigen-induced AHR, and that the DBA/2alleles, in aggregate, functioned in a dominant manner to limit IL-4production and AHR. These results underscore the multigenic, complexnature of atopic traits and demonstrate the potential advantages ofusing a congenic strain to isolate and characterize a single locuswithout interference from multiple epistatic genes that also influencethe asthmatic phenotype.

Genetic Mapping of Tapr, the locus which controls AHR and IL-4responsiveness. Previously, the congenic region in the HBA mice wasdelineated with 36 genome-wide markers, including two chromosome 11markers, hemoglobin-α2 (hba-α2) and esterase-3 (es-3) loci. The HBAgenome, outside of chromosome 11, was inherited from BALB/c. A moreprecise analysis with 25 simple sequence length polymorphism (SSLP)markers known to be polymorphic between DBA/2 and BALB/c mice showedthat HBA mice inherited two segments of chromosome 11 from DBA/2 (FIG.2, left column). The proximal region contained a 20 cM segment withhomology to chromosome 5q23-35, which afforded the possibility that agenetic locus implicated in human asthma linkage studies could beidentified in a mouse model of asthma.

To map at higher resolution the T_(H)2-AHR controlling locus, Tapr,(BALB/c×HBA) FI mice were backcrossed to HBA mice to produce N2 animals.With this backcross approach, the set of alleles contributed by the HBAparent is pre-determined, and the set of alleles contributed by the F1parent can be assessed by genotyping. Thus, recombination events thatproduce informative haplotypes within the congenic region can bedetected in the N2 mice and used to assess the linkage of Tapr to lociin the congenic interval. Because of the recessive nature of Tapr, wetested N2 mice from these backcrosses to identify the minimum homozygousregion of HBA-derived genes sufficient to confer the HBA Tapr phenotype.More than 2,000 N2 animals were generated and genotyped. Using SSLPmarkers, we selected those N2 mice with informative recombinationevents, and the N2 mice were phenotyped for the capacity to produce IL-4in response to immunization with keyhole limpet hemocyanin (KLH). Inthis primary analysis, we determined that the relevant locus residedwithin the proximal congenic region, between D11Mit135 and D11Mit260. Inorder to map Tapr at higher resolution, 22 additional markers wereidentified and utilized to provide 0.1-1 cM resolution in the area ofinterest.

To accurately compare the results of IL-4 cytokine analyses performedover several months time, an IL-4 index for each experiment wasgenerated for each N2 mouse,

$\left( \frac{B - x}{B - H} \right),$where B=IL-4 production by cells from BALB/c mice, H=IL-4 production bycells from HBA mice, and x=IL-4 production by cells from the N2 mousebeing assessed. High concentrations of IL-4 (BALB/c-like) arerepresented by index values near 0, and low concentrations of IL-4(HBA-like) are represented by index values near 1.0. The “B and “H”values were established with 3-5 control mice for each group of 3-6 N2mice carrying informative recombinations that we tested. The indexvalues fall within a bimodal distribution (FIG. 3 a), in which thephenotype index associated with N2 mice that had nonrecombinant HBAgenotypes was significantly higher (P<0.0001, in a paired Student'st-test) than the phenotype index associated with N2 mice that hadnonrecombinant (BALB/c×HBA)F1 genotypes.

For the mice with unique genotypes, we used several methods to ensurethe adequacy of single measurements of cytokine production and AHR,since this is critical in linkage analysis. First, at the same time thatwe tested each of the N2 mice carrying recombinations of interest, wealso tested “non-recombinant” siblings of each “recombinant N2” thatwere strictly HBA or F1 (BALB×HBA) in genotype. Furthermore, we bredadditional N3 mice by crossing some of the N2 mice carryingrecombinations of interest back to HBA mice, in order to have moreindividual mice with that particular N2 genotype. All values were theaverage of the values for the individual mice tested with a givengenotype. In this way, we are confident of the measures of cytokineproduction and AHR, and that we have overcome assay variations due tovariables inherent in biological systems.

Because the IL-4 values associated with the N2 mice that inheritedrecombinant haplotypes segregated in a bimodal distribution (FIG. 3 a),were able to demonstrate that the genetic locus that controls high IL-4responses is located between markers D11Mit271 and D11Mit22 (FIG. 3 b).Moreover, high levels of IL-4 production were observed in all mice witha BALB/c allele present at Kim1sscp, and low levels of IL-4 productionwere observed in all mice with homozygous HBA genotypes at Kim1sscp.Thus, Tapr was nonrecombinant with Kim1sscp, an intronic marker within amouse homologue of Rattus norvegicus Kidney Injury Molecule (Kim-1). Incontrast, Tapr segregated from all other markers with at least onerecombination. The fact that Tapr and Kim1sscp segregated together,indicated that the Tapr locus is located very close to or isindistinguishable from Kim1sscp. Based on the frequency recombinanthaplotypes between D11Mit271 and D11Mit22, we calculate a recombinationfrequency, 0.0039, which indicates that that the Tapr locus maps to asmall, 0.3-0.5 cM, region. We also calculated a recombination frequencyof 0.08 between Tapr and IL-4. Therefore, Tapr is located 5-10 cM awayfrom the IL-4 cytokine cluster but is within the a region of the mousegenome that has highly conserved synteny with the 5q23-35 region thathas been linked to human atopy and asthma.

Using an analogous approach, we examined the segregation ofallergen-induced AHR phenotypes in mice with informative recombinanthaplotypes. With indexed AHR values, N2 mice clearly exhibit parentalphenotypes, which produced a bimodal distribution in a histogram of AHRindex values in a group of sensitized N2 mice (FIG. 3 c). By analyzingthe segregation of AHR phenotypes associated with more than 1,000 N2mice, we demonstrated that the genetic locus which controls AHRresponses is also located between markers D11Mit271 and D11Mit22 (FIG. 3d) and that the AHR phenotype was nonrecombinant with Kim1sscp. Thus, wedemonstrate that both IL-4 responsiveness and AHR cosegregate with theTapr locus, which suggests that the same locus regulates both IL-4expression and AHR (FIG. 3).

These findings further demonstrate that the Tapr locus is more than 5 cMcentromeric to the IL-4 cytokine cluster and the cytokine genes in thecluster previously thought to be ‘candidate’ atopy or asthmasusceptibility genes. Our mapping results also establish that Tapr isgenetically separable from both the IL-12p40 gene and the region ofmouse chromosome 11 that includes the T_(H)1-IL12 regulatory locus, Tpm.

Mouse and human homologues anchor Tapr to human 5q33. In order toconstruct a composite map around the Tapr locus, we integrated availableinformation from the Mouse Genome Database (MGD) linkage, backcross, andradiation hybrid maps and identified a region of conserved synteny inmaps of the human genome. Current radiation hybrid maps place themarkers that are near D11Mit271 and D11Mit22, including severalexpressed sequence tags (ESTs) that have extensive homology to knowngenes or unigene clusters, onto a physical map of the mouse genome. Wefurther examined these markers and their associated ESTs for previouslyunidentified similarity to known gene clusters. We assembled thesemarkers onto a scaffold for comparison to the human genome. Using thisapproach, we found significant similarity between particular radiationhybrid markers and the following human genes: KIAA0171, Adam-19, Sox-30,Pir-121, Crsp9 (Crsp33), and hHAVcr-1 (hHAVcr-1). FIG. 4 demonstratesthat once we anchored these genes to a physical map of the mouse genomebetween our flanking markers, we were able to locate those genes in theHuman Genome Browser.

The high degree of conservation between the mouse and human genomes inthis region indicates linkage of the Tapr locus to human 5q33.2. Asshown FIG. 4, we identified all known genes and ESTs in this region ofthe human map. Genes of particular interest near human hHAV-cr and themouse homologue of Kim-1, include IL-2 inducible T cell kinase (Itk) anda coregulator of the SP-1 transcription factor (Crsp9), both known to beinvolved in T cell differentiation. We sequenced coding regions fromthese candidate genes and found no polymorphisms in either ITK orCRSP-9.

Localization of a Family of Novel T cell Surface Proteins to the TaprRegion. Because the mouse homolog of rat Kim1 is located within the 0.4cM region and is tightly linked with Tapr, we examined publiclyavailable databases and found clusters of ESTs with some sequencesimilarity that provided only partial coverage and contained largesegments of variation. The closest human homolog of Kim-1 is the humanhepatitis A virus cellular receptor, hHAV-cr, and tBLAST searches of thehuman genome suggested that two additional homologs of Kim-1, perhapsmembers of a gene family, also are located on human chromosome 5 andmouse chromosome 11.

Using cDNA from conA-stimulated splenocytes, we identified and clonedtwo mouse orthologues of Kim1, which we term Tim1 and Tim2, that map tothe Tapr region, as shown in FIG. 5A. TIM-3 is a third, more distantlyrelated, orthologue of KIM-1.

All three members of this gene family are expressed by stimulated Tcells, and all three forms map to the Tapr region of mouse chromosome11/human chromosome 5 where they encode cell surface glycoproteins withcommon structural motifs, including an immunoglobulin (Ig) domain, mucindomain, and intracellular tail with phosphorylation sites. Because thecellular functions of these proteins is unknown, we refer to the genesas members of a T cell, Immunoglobulin domain, Mucin domain (Tim) genefamily. Mouse Tim1 is the mouse homologue of rat Kim1 and the HAVcr-1identified in African green monkeys and humans. Tim2 is a previouslyunknown gene that had not been identified in any organism prior to thisstudy.

The mouse Tim1 gene encodes a 305 amino acid membrane protein, that has78% overall identity with rat KIM-1 and 35% identity with human HAVcr-1.A gapped multiple sequence alignment with mouse TIM-1, rat KIM-1, humanHAVcr-1 and African green monkey HAVcr-1, shown in FIG. 5B, demonstratesthe degree of homology between the TIM-1/KIM-1/HAVcr-1 proteins in thesespecies. The cytoplasmic region of TIM-1 contains two tyrosine residuesand includes a highly conserved tyrosine kinase phosphorylation motif,(SEQ ID NO:59) RAEDNIY, which is integral to the predicted Itk and EGFRkinase site of TIM-1, (SEQ ID NO:601 SRAEDNIYIVEDRP. The mucin domain ofTIM-1 has multiple sites for O-linked glycosylation, and there two sitesfor N-linked glycosylation found in the immunoglobulin domain.

TIM-2, a similar 305 amino acid membrane protein, has 64% identity tomouse TIM-1, 60% identity to rat KIM-1, and 32% identity to hHAVcr-1(FIG. 5A, B). Like TIM-1, TIM-2 has two extracellular N-linkedglycosylation sites and a serine, threonine-rich mucin domain with manyO-linked glycosylation sites. TIM-2 also has an intracellular tyrosinekinase phosphorylation motif, RTRCEDQVY.

Tim3 encodes a 281 amino acid membrane protein that has a similar,integral membrane glycoprotein structure with multiple extracellularglycosylation sites and an intracellular tyrosine phosphorylation motif.Although the mucin domain is not as prominent in TIM-3 as it is in TIM-1and TIM-2 (FIG. 5A), TIM-3 expressed on T cells likely interacts with aligand on APCs and alters APC activation. TIM-3 does have four sites forN-linked and five sites for O-linked glycosylation, suggesting thatTIM-3, like TIM-1 and TIM-2, is heavily glycosylated and might interactwith a ligand present on other cells, such as antigen presenting cells.

Tim4 encodes a 344 amino acid protein in mice, and a 378 amino acidprotein in humans. The predicted TIM-4 also shares the general membraneglycoprotein structural motifs of the other TIM proteins, a with anIgV-like domain with highly conserved cysteine residues, athreonine-rich mucin-like domain, and a short intracellular tail.However, TIM-4 lacks the phosphotyrosine motif present in the other TIMproteins, and therefore may modulate the funtion of the other TIMproteins.

Each of the TIM Ig domain shares an predicted integrin-binding motifthat is similar to the SVVYGLR motif found in osteopontin, antransmembrane protein like the TIMs that is implicated in the regulationof cell adhesion, survival, and oncogenesis, as well as in theregulation of helper T cell differentiation. This integrin binding motifdemonstrates alpha(9) and alpha(4) specificity.

Comparison of the sequences of the BALB/c and HBA/DBA coding regions forthe three Tim genes revealed major polymorphisms in TIM-1, TIM-3, andTIM-4, but not TIM-2. In TIM-1, these polymorphisms encode three aminoacid differences and a fifteen amino acid deletion in HBA/DBA. Sevenpredicted amino acid differences were identified in TIM-3 (FIG. 5 c).Genomic sequences confirm that these polymorphisms, including thedeletion, are true polymorphisms, not splicing variants. By furthersequencing genomic segments of TIM-1 and TIM-3 in other mouse strains,we found that C57/BL6, a strain similar to DBA/2 with respect to itstendency to develop reduced T_(H)2 and AHR responses, also has theHBA/DBA allele of Tim1 and Tim3. The polymorphisms in TIM-1 and TIM-4are located in the signal and mucin-like domains, while thepolymorphisms identified in TIM-3 are clustered in the Ig domain (FIG. 5c). In glycoproteins with Ig and mucin domains, variants in eitherdomain may affect receptor-ligand interactions, as shown for MAdCAM-1.Although the predicted cleavage sites of TIM-1 and TIM-4 are unalteredby the polymorphism in the signal sequence, it is possible that thepolymorphism may affect the efficiency of cleavage and/or trafficking ofthe receptor to the cell surface. These Tim sequences and polymorphismsare important for immune responses, and for HAV viral pathogenesis inhumans.

Analysis of genomic DNA samples from our N2 backcross (FIG. 3)demonstrated that the TIM-1 and TIM-3 polymorphisms cosegregatecompletely with Tapr. While these observations do not distinguish theextent to which changes in TIM-1, TIM-3, or both, are responsible forchanges in AHR and T_(H)2-mediated inflammation, we suggest thatpolymorphisms in human TIM-1(hHAVcr-1) and/or TIM-3 underlie the strongassociation between asthma susceptibility and human chromosome 5q. Thisidea is supported by the fact that major variants in coding regions ofhuman Tim1 are evident on examination of human genome and EST databases.Comparison of these human cDNA variants with the previously describedvariants of monkey HAVcr-1 and the mouse variants identified heredemonstrates that there is extensive variation in the predicted proteinsequences of TIM-1 (FIG. 5 b,c). This high degree of variationdistinguishes TIM-1 and its family members from many other candidategenes, such as the cytokines and the cytokine receptors that have beenmost closely studied as asthma susceptibility candidate genes. Inaddition, the association between Tim1 and asthma susceptibility isfurther supported by reports of significant linkage of mite-sensitivechildhood asthma to D5S820 (mean LOD score=4.8), a marker which isapproximately 0.5 megabases from Tim1 and Tim3 (FIG. 4.

In addition to the above genetic polymorphisms, there are severalexpression polymorphisms in the TIM genes that arise due to alternatesplicing. Alternate splicing of TIM-1, TIM-2 and TIM-4 mRNA producesseveral TIM variants, some of which are secreted, soluble forms of theTIM receptors. These splice variants, along with TIM splice variantsthat have alternate 5′ untranslated regions, may contribute to thecell-specific and condition-specific expression patterns of the TIMproteins.

T cells confer the Tapr effect. To better understand the function of theTapr locus we determined whether allelic variation of Tapr affected thefunction of T cells or of antigen presenting cells (APC). For theseexperiments, we generated ovalbumin (OVA)-specific T cell receptor (TCR)transgenic mice (Tg) with the HBA background (HBA DO11.10), which wecompared to TCR-Tg mice with the BALB/c background (BALB/c DO11.10).Purified CD4+ T cells from either of these strains were cocultured withOVA and dendritic cells (DCs) derived from either BALB/c or HBA bonemarrow, and the cytokines produced were evaluated. Irradiated spleencells were not used as APCs for this experiment, because it was foundthat irradiated spleen cells and other tissues express high levels ofthe TIM genes.

BALB/c DO11.10 T cells produced higher levels of IL-4 and IL-13 than didHBA DO11.10 T cells, in a manner that was independent of the source ofthe antigen presenting cells (FIG. 6A). In addition, the source of theCD4 T cells determined the amount of IL-4/IL-13 produced at each antigenconcentration, regardless of the source of the APC during either theprimary or secondary stimulation. Equivalent levels of IL-12 weredetected in culture supernatants for each combination of cell types,further demonstrating that BALB/c and HBA DC function were comparable.Furthermore, BALB DO11.10 and HBA DO11.10 T cells produced equivalentlevels of IL-2 and demonstrated comparable levels of proliferation inresponse to OVA during the secondary cultures, indicating that HBA andBALB/c T cells are similarly activated, although the levels of Th2cytokines they produce are quite distinct.

We show in FIG. 6B that within the first twelve hours of primary culturein our DO11.10/DC system, we find that mRNA for TIM-1 is expressed byboth BALB/c and HBA CD4+ T cells. Within four days of primarystimulation, we find significant levels of IL-13 in supernatants of theBALB/c DO11.10 and detect none in the HBA DO11.10 supernatants. Thisdifferentiation is detectable in mRNA levels at 36 hours (FIG. 6B).Between twelve and thirty six hours, expression of IL-13 mRNA is reducedin HBA CD4 T cells, while IL-13 expression is maintained in the BALB/cCD4 T cells. Thus, during the primary response to antigen, BALB/c CD4 Tcells develop a stronger Th2 response than do HBA CD4 T cells. Ourfindings demonstrate that Tapr regulates helper T cell differentiationduring primary antigen specific responses, and we detect TIM-1expression in CD4 T cells during the earliest stages of these responses.

Following differentiation into mature Th1 and Th2 subsets, helper Tcells demonstrate committed TIM expression by RT-PCR, such that Th1cells express TIM-3, while Th2 cells preferentially express TIM-1. All Tcell populations demonstrate weak TIM-4 expression. While the Itk signalthrough TIM-1 is likely to promote Th2 differentiation, the EGFR signalthrough the TIM proteins is likely to enhance cell survival in effectorand especially memory T cell populations. Since Itk is expressed only inT cell and mast cells, the Itk kinase activity on TIM-1 is restricted toimmune cells, particularly those involved in asthma and allergy.However, other protein tyrosine kinases, such as EGFR, are involved inthe function of TIM proteins expressed by other tissues, includingishemic epithelial cells, irradiated spleen cells, and tumor cells.

In these studies, we mapped Tapr, a locus that regulates the developmentof Th2 cytokine production and antigen-induced AHR, a cardinal featureof asthma. We localized Tapr using an interval specific congenic mouse(HBA) that carried a chromosomal segment homologous to human chromosome5q, a region of the human genome that has been repeatedly linked toatopy and asthma. This region has also been repeatedly linked to5q-syndrome associated with myelodysplasia and neoplastic cytogenicabnormalities, Using this congenic mouse strategy that converted acomplex trait into a simpler, possibly single gene, trait, we narrowedthe interval of Tapr to 0.4 cM interval, sequenced several candidategenes in this region, and positionally cloned the TIM gene family.

The TIM gene family has not been previously described. We identified andcloned the full cDNA sequence and discovered significant polymorphismsin the TIM-1 proteins of BALB/c compared to HBA mice. We found that theBALB/c sequences for TIM-1 and TIM-3 are associated with susceptibilityto AHR and allergic T cell responses, whereas the HBA sequences areassociated with protection against these responses. TIM-3 ispreferentially expressed by differentiated T_(H)1. The association ofpolymorphic Tim3 variants with Tapr suggests that TIM-3 might regulateT_(H)1 and T_(H)2 cell function. However, the variations in Tim3 mightalso be attributed to a haplotype tightly linked to Tim4 or Tim1.

We believe that TIM-1 plays a very important role in the regulation ofthe immune system (particularly with respect to asthma and allergicdisease) and in the regulation of epithelial and hematopoetic cellsurvival in response to stress (hypoxia, nutritional deficiency,irradiation, chemotherapy, etc.) for several reasons. First, Tim1, likeTim3, is expressed in CD4 T cells during primary antigen stimulation,when it is most likely that the Tapr effect occurs. T cells play acritical role in the development of AHR and in the pathogenesis ofasthma, our results suggest that Tapr affects asthma by enhancing earlyCD4 commitment to Th2 responses by controlling the production of IL-13and subsequent T cell differentiation. Second, HAV infection in humansduring infancy or childhood is inversely associated with the developmentof asthma and allergy. We suggest that the HAV interaction withTIM-1/HAVcr-1 may alter the T cell cytokine production may able toreverse or prevent the biased Th1/Th2 balance in individuals otherwiseprone to atopy and asthma. SLAM, a measles virus receptor, is an exampleof another T cell surface glycoprotein that regulates the Th1/Th2balance in a manner that may be altered by viral interaction. Becausesome viral receptors, such as SLAM for the measles virus or CD4, CCR5,and CXCR4 for HIV, are receptors of the host's own immune system, evenwhen an infection does not succeed, virus-receptor mediated signaltransduction can lead to the release of cytokines and the development ofdisease.

Third, the polymorphisms in TIM-1 are associated with the differenttypes of helper T cell responses that we observe. Therefore, thevariants of TIM-1 may themselves contribute to the genetic Th1/Th2predisposition that occurs in the absence of any known environmentalcause of immune deviation. The HAV receptor in primates is known to behighly variable, and we propose that polymorphic alleles of humanTIM-1/hHAVcr-1, like those we have identified in mice, may be associatedwith variations in Th2 bias and asthma susceptibility. Mutations in thegenes for cell surface molecules that serve as viral receptors and thatalter susceptibility to infection are not uncommon, and thereforesignificant genetic variation in TIM-1 and other members of the TIM genefamily is far more likely to be observed than variation in other genessuch as those for cytokines. It is unclear why asthma susceptibilityalleles might be prevalent in the human gene pool, but the associationof Tapr with HAVcr provides an interesting explanation for thepersistence of asthma susceptibility alleles. During human evolutioncertain alleles of the Tim gene family may have conferred resistance toatopic diseases and other immune disorders, but selection of thoseresistance alleles may have been counterbalanced by selection ofalternate alleles that confer resistance to viral pathogenesis.

In summary, our studies represent the first successful utilization of acongenic mouse strategy to locate a strong candidate asthmasusceptibility gene and overcome the inherent difficulties in theexamination of this complex genetic trait. We identified a previouslyunknown gene family that exists in a region homologous to humanchromosome 5q, and which plays a major role in Th cell development andin asthma susceptibility. While prior studies in humans identifiedseveral candidate genes on human chromosome 5q, the Tim1 gene productidentified in our study also provides an explanation for the inverserelationship between HAV infection and reduced asthma susceptibility.

Subpopulations of CD4⁺ T cells (Th) produce distinct patterns ofcytokines, and this has led to the concept of functional heterogeneityamong Th cells. Type 1 Th cells (Th1) produce interleukin 2 (IL-2)and/or interferon γ, elicit delayed type hypersensitivity (DTH)responses and activate macrophages. Type 2 Th cells (Th2), on the otherhand, produce IL-4, IL-5 and IL-10 and are especially important for IgEproduction and eosinophilic inflammation, and may suppress cell mediatedimmunity. Th2 cells are believed to play a pivotal role in thepathogenesis of atopy. Several factors determine whether a T helper cellwill differentiate into Th1 versus Th2 during a particular immuneresponse. These include, but are not necessarily restricted to, thecytokine milieu, the strength of the TCR signal and/or antigen density,and the costimulatory pathways. CD4+ T helper cell differentiation intoTh1 or Th2 subsets has profound effects on the outcome of atopy,autoimmune diseases, infectious diseases, and graft rejection.

The specific features of immune responses that protect nonatopicindividuals from the development of allergic diseases and which couldinhibit allergic responses in atopic individuals are poorly understood.Because Th1 cells cross regulate Th2 cells in some systems,allergen-specific Th1 cells have been assumed to regulate allergicdisease and asthma. Th1 cells inhibit the development and proliferationof Th2 cells, and IgE production is reciprocally regulated by IL-4 andIFN-γ. This suggests that protection from allergy is due to thedevelopment of inhibitory allergen-specific Th1 cells. Allergen-specificT cell clones derived from the peripheral blood of nonallergicindividuals have been shown to produce Th1 cytokines. These observationshave also supported the hygiene hypothesis of asthma, which suggeststhat the prevalence of infections, particularly those that induce Th1responses, are reduced in westernized societies by improved publichealth measures and the use of vaccines and antibiotics. As a result,Th2 responses and atopy develop more intensely and rapidly in theabsence of Th1 mediated responses.

The TIM genes identified herein are also candidate oncogenes.Transfection of cell lines with TIM genes confers resistance to celldeath, and the predicted EGFR kinase motif described in TIM-1 provides aprobable mechanism by which this cell survival is controlled.Furthermore, TIM-1 demonstrates a significant degree of sequenceidentity (approximately 20%) and structural similarity (a transmembraneglycoprotein with an IgV domain, mucin/syndecan domain, transmembranedomain, and intracellular domain with similar phosphotyrosine motifs)with TOSO, a protein that protects cells from Fas-mediated apoptosis.Like the TIM genes, TOSO is a likely oncogene, which maps to a region ofthe genome with frequent changes in hematologic malignancies and solidtumors.

Methods

Animals.

Congenic lines, including C.D2 Es-HBA were generated by introgressivelybackcrossing DBA/2N onto a BALB/cAnPt background. BALB/cBy, DBA/2J, and(BALB/c×DBA/2) F1 mice (CByD2F1/J) were obtained from the JacksonLaboratory (Bar Harbor, Me.), while BALB/cAn and DBA/2N were obtainedfrom Taconic Labs. (BALB/c×HBA) F1 mice were produced with a crossbetween BALB/cByJ and HBA. N2 mice were generated by backcrossing(BALB/c×HBA) F1 to HBA. In our analysis of recombinant N2 animals,recombinant mice were tested along with non-recombinant siblings,whenever possible. In order to examine individual N2 genotypes inmultiple assays, we preserved selected recombinant haplotypes bybackcrossing selected N2 mice to HBA to generate N3 mice, which weregenotyped to chose mice carrying the recombinant N2 haplotype. DO11.10mice, which are transgenic for TCR recognizing OVA peptide 323-339(pOVA³²³⁻³³⁹) and backcrossed to BALB/c(43), were kindly provided by Dr.Dennis Loh and were bred in our facilities. HBA DO11.10 mice wereproduced by backcrossing DO11.10 to HBA. DO11.10 mice were selected byFACS analysis for the TCR-Tg and genotyped to select for HBA allelesbetween D11Mit135 and D11Mit168. The Stanford University Committee onAnimal Welfare approved all animal protocols.

Genotypinq.

Additional markers around the Tapr locus were identified by testing allavailable “D11Mit-” markers present between D11Mit140 and D11Mit269 andall radiation hybrid markers near D11Mit271 and D11Mit22 for anypolymorphisms between DBA/2 and BALB/c. MIT MapPair primers wereobtained from Research Genetics (Huntsville, Ala.), and all otherprimers were synthesized in the Protein and Nucleic Acid Facility(Stanford, Calif.). PCR was performed as previously described, and SSLPpolymorphisms were resolved with 4-5% Metaphor agarose (BioWhittaker,Walkersville, Md.). Products analyzed for SSCP were amplified with³³P-dCTP and separated on denaturing acrylamide gels at 40 W and 4²C,with a Sequi-Gen GT System (Bio-Rad, Hercules, Calif.).

Immunization Protocols.

Mice studied in cytokine production assays were primed with KLH(Calbiochem, La Jolla, Calif.) in complete Freund's adjuvant (CFA)(DeKruyff et al. J Immunol 149, 3468-76 (1992)). For measurement ofairway hyperreactivity, mice were immunized with OVA intraperitoneally(i.p., 50 μg) complexed with aluminum potassium sulfate (alum) on day 0,and intranasally (i.n. 50 μg OVA in 50 μA of PBS) after light anesthesiaon days 7, 8 and 9. Control mice received i.p. injections of alum aloneand intranasal PBS. Airway hyperreactivity to inhaled methacholine wasmeasured 24 hours after the last intranasal dose of OVA (day 10).

Measurement of Airway Responsiveness.

Airway responses were assessed by methacholine-induced airflowobstruction from conscious mice placed in a whole body plethysmograph(Buxco Electronics Inc., Troy, N.Y.), as described previously (Hansen etal. J Clin Invest 103, 175-83 (1999)).

Cell Culture.

Lymph node cells from mice primed with KLH were prepared as describedpreviously (Yeung et al. J Immunol 161, 4146-52 (1998)). TransgenicDO11.10 CD4 T cells were positively selected using MACS columnsfollowing incubation with anti-CD4 magnetic beads (Miltenyi Biotech,Germany). 2×10⁴ cells/well were cocultured in 96-well round bottomplates with 250 μg/ml OVA and 1×10⁴ bone marrow-derived dendriticcells/well. After seven days, the DO11.10 T cells were washed andrestimulated with fresh antigen presenting cells and antigen at theconcentration indicated. Antigen concentration for the primary DO11.10cultures was titrated during the restimulation. Bone marrow-deriveddendritic cells were generated as previously described with somemodifications; 5×10⁶ bone marrow cells were cultured in 9-cm diametertissue culture dishes with 10 ml culture medium containing 20-25 U/mlGM-CSF. Loosely adherent cells were transferred onto a second dish onthe sixth day of culture; within four days, these transferred cells wereused as a source of dendritic cells.

Cytokine ELLISA.

ELISAs were performed as previously described in Macaulay et al. JImmunol 160, 1694-700 (1998); and Macaulay et al. J Immunol 158, 4171-9(1997).

Monoclonal Antibodies.

Monoclonal antibodies for ELISA and FACS analysis were purified fromascites fluid by ammonium sulfate precipitation and ion-exchangechromatography. Anti-clonotypic antibody KJ1-26.1, was generouslyprovided by Dr. Philippa Marrack, National Jewish Medical Center, andthe antibody was FITC-conjugated according to standard protocols priorto FACS.

Example 2 Identification of Human Tim Sequences

The positional cloning of the TIM gene family within a locus thatconfers protection against the development of Th2 responses andallergen-induced airway hyperreactivity provides an opportunity togreatly improve our understanding of the regulation of Th2 drivenresponses and atopic diseases. In addition, TIM-3 is specificallyexpressed on murine Th1 cells and anti-TIM-3 mAb leads to increasedseverity of experimental autoimmune encephalomyelitis (EAE). Thisemphasizes the importance of the gene family in T helper subsetregulation.

The human Tim cDNAs, which are the orthologues of murine Tim-3 and Tim-4were cloned by PCR. The human orthologue of TIM-1 was cloned as HAVcr-1,the cellular receptor for hepatitis A virus. The TIM family genes areimmediately adjacent to each other on human chromosome 5, in the orderTIM-4, TIM-1, TIM-3, with no intervening genes. There are TIMpseudogenes on chromosomes 12 and 19. The gene family members are onlymoderately related. The protein sequences and relationship among the Timgene family are shown in FIG. 7.

The cytoplasmic domains of TIM gene family members are the mostconserved domain between mouse and human orthologues, e.g., 77% identitybetween the human and mouse TIM-3 cytoplasmic domains. In contrast, thewhole TIM-3 is only 63% identical between human and mouse. Each TIM genecontains a distinct predicted tyrosine signaling motif. The cytoplasmicregion of TIM-1 contains two tyrosine residues and includes a highlyconserved tyrosine kinase phosphorylation motif, (SEQ ID NO:59) RAEDNIY.The expanded region, (SEQ ID NO:60) SRAEDNIYIVEDRP, contains a predictedsite for Itk and EGF receptor phosphorylation. Itk is known tophosphorylate phospholipase C-γ (PLC-γ), and thereby trigger a cascadeof signaling events that are involved in T cell activation and helper Tcell differentiation. Furthermore, Itk signaling affects Th1/Th2differentiation, and Itk^(−/−) mice do not develop strong Th2 responses.EGF receptor kinase activity is associated with cell survival andresistance to cell death. Similarly, TIM-3 contains distinct, conservedtyrosine phosphorylation and SH2 binding motifs in the cytoplasmicdomain. This suggests that the interaction of a TIM with its ligand willengage an intracellular signaling pathway and that each TIM will bedistinct in this signaling.

The extracellular IgV domain of the TIM proteins also contains apredicted integrin-binding motif that is similar to the SVVYGLR motif ofosteopontin that is involved in adhesion via alpha(9)beta(1),alpha(4)beta(1), and alpha(4)beta(7) integrins. TIM-1 transfected pre-Bcells of the 300.19 line demonstrate a high degree of adhesion anincreased survival in cell culture, as compared to non-transfected300.19 cells. TIM-1 and TIM-2 transfected CHO cells also demonstrateenhanced survival compared to untransfected CHO cells. These resultsdemonstrate that the TIM proteins regulate cell adhesion and death

Genetic Polymorphisms in the Human Tim1 and Tim3 Genes.

SNPs or nucleotide polymorphisms and deletions/insertions present in thehuman Tim1 gene are identified. Because SNPs are extremely common in thegenome, occurring every 300-600 base pairs, only the coding region ofTim1 was analyzed. Moreover, genetic variations that are common are alsolikely to be important. Initially cDNA is sequenced from T cells takenfrom 30-40 individuals (60-80 chromosomes). Power calculations show thatsurveying target sequences in coding regions of 60 chromosomes willeasily detect SNPs with a population frequency of greater than 1%, andhaving a more than 90% chance of detecting alleles with a populationfrequency of 5% or greater. Therefore, screening 30-40 individuals forsequence variations captures most of the common, functionally relevant,non-conservative, DNA variation present in a population.

Since DNA variants/SNPs in close physical proximity often show strongdependency relationships (i.e., linkage disequilibrium), it isdetermined if a group of DNA variants (SNP haplotypes) are inheritedtogether, and determined if screening for only a portion of these SNPswill be sufficient for identifying the haplotype. Analysis of largeregions of various chromosomes indicate that discrete haplotype blocks(of tens to hundreds of kilobases) are generally present, each withlimited diversity punctuated by apparent sites of recombination. To findhaplotypes, cDNA is sequenced and searched for combinations of sequencevariations that are seen repeatedly in multiple individuals. Peripheralblood mononuclear cells (PBMC) were from 38 donors, and were stimulatedin vitro with PHA (7.5 μg/ml) for 24 and 72 hours, or with Concavalin A(2 μg/ml) for 24 hours. PMA (20 ng/ml) and lonomycin (1 μM) were addedduring the last six hours of stimulation. The cells were then harvestedand the total RNA was extracted using Trizol reagent (Invitrogen). Toobtain cDNA templates for sequencing, RNA was reverse transcribed usingSuperscript II reverse transcriptase (Invitrogen), according to themanufacturer's protocol. The cDNA were used to PCR amplify the fulllength of Tim cDNA using Herculase Hot Start™ high fidelity polymerase(Stratagene). The PCR primers were: (SEQ ID NO:41) GTGTCTGACAGTGGCGTA(forward), (SEQ ID NO:42) TTTGCCCAGGCAGAACCA (forward), (SEQ ID NO:43)CCACCCAAGGTCACGACT (reverse), (SEQ ID NO:44) ATGCCACGGACTAAGACC(reverse). The PCR products were purified with. Qiagen QIAquick gelextraction reagents, and sequenced using four internal sequencingprimers for Tim1 and two internal sequencing primers for Tim3.

The full length Tim1 RT-PCR product was cloned in these individuals bytaking total RNA from activated T cells and transcribing it withSuperscript II and oligo dT. Tim1 cDNA was amplified with Expand highfidelity polymerase (Roche) to generate a 1 kb product spanning the Tim1coding region, which was purified with a PCR Purification kit(Invitrogen). This purified product was then cloned into the TOPO pEF6vector (Invitrogen), followed by transformation of TOP10 competentbacteria. Bacterial colonies were grown on LB plates with ampicillinselection. Single colonies were picked and plasmid preps generated usingQiagen mini prep kits. Restriction mapping using Hind III digestion wasused to select plasmids containing inserts in the correct orientation.These plasmids were then sequenced with three different primers, forward(T7), internal and reverse (BGH), and the sequences aligned in SeqManprogram with NCBI human TIM reference sequence.

After sequencing Tim1 from the chromosomes from 35 individuals (70chromosomes) several polymorphisms in Tim1 were identified, which areshown in FIG. 8. These polymorphisms are numbered 1-7 (left column). Thefull sequence of human TIM-1, which is listed in the NCBI database(NM_(—)012206), is provided in FIG. 8 as a reference point. Thissequence is present in less than 20% of the chromosomes that weresequenced, due to the existence of multiple, prevalent sequencepolymorphisms in the coding region. 6 additional sequence variationswere identified, shown in FIG. 8, and all of the polymorphisms wereobserved in the mucin, extracellular domain, as was true for mice,although the specific variations were distinct from those seen in mice.Importantly, there is a limited degree of association between thesevariants, in various combinations. The most pronounced variations arethe insertion labeled polymorphism 1, 157insMTTTVP (SEQ ID NO:57), whichwas observed in 65% of the chromosomes, and the deletion in polymorphism5, 187ΔThr, was observed in 65% of the chromosomes. Polymorphism 4 wasobserved in 40% of the chromosomes, and the other polymorphisms wereeach observed in ≦5% of the chromosomes. Notably, most of thesevariations (2-6) are located within exon 3, the first mucin-encodingexon, and all of the variants occur at the genomic level and are notsplice variants.

Based on this sequence analysis of mRNA, a more rapid method foranalyzing the genomic DNA from the larger number of patients/controlshas been developed. To screen individuals for the variations seen insequences shown in FIG. 8, the DNA is initially tested for simplesequence length polymorphisms (SSLP) in a 150 PCR product, which candetect the major insertion, polymorphism 1, and the deletion,polymorphism 5.

In addition, to genotype the other polymorphisms (2-4, 6, and 7) andidentify novel polymorphisms, a relatively simple assay using singlestrand conformational polymorphism (SSCP) analysis of PCR products hasbeen developed. Under well-optimized conditions, SSCP analysis detectsmore than 90% of single nucleotide substitutions and all lengthpolymorphisms. For this analysis, PCR primers have been identified thatamplify each exon of the Tim genes, and variants can be distinguishedusing standard non-denaturing SSCP gel electrophoresis methods (FIG. 9).Non-denaturing polyacrylamide gel electrophoresis is used with an ABI377 DNA sequence for high resolution SSCP analysis of each exon.Fluorescent end-labeled primers are synthesized and purified. Novel SSCPpatterns that are detected during the high-throughput genotyping processwill identify novel variants. Using this method, the genotype ofpatients and controls is rapidly analyzed.

The Tim3 gene was analyzed using essentially the same methodologies.mRNA from activated T cells is sequenced to identify Tim3 polymorphisms,as well as long range haplotypes between the Tim1 and Tim3. Aftersequencing Tim3 cDNA representing 60 chromosomes, it has been found thatTim3 is polymorphic, as it is in the mouse genome. However, only onepolymorphism, Leu140Arg, is prevalent, found in approximately 12% of thechromosomes represented.

Example 3 Expression of Tim Sequences

Murine TIM-3 protein is expressed on Th1 clones but not on naive T cellsor Th2 cells. Using TCR transgenic T cells, TIM-3 protein was notexpressed on Th1 cells after one or two rounds of Th1-directeddifferentiation but was expressed after the third and further rounds ofTh1 stimulation. TIM-3 mRNA expression was detected somewhat earlier. Inorder to determine if TIM-3 gene expression was the same in human, TIM-3and TIM-1 mRNA expression in human Th1 cells was examined using tetanustoxoid specific T cells generated by stimulation with antigen in thepresence of IL-12 and anti IL-4 mAb. Given the association of TIM-1 withasthma, TIM-1 and TIM-3 mRNA expression in human Th2 cells was examined.Th2 cell lines were generated from allergic donors by in vitrostimulation with allergen, IL-4, and anti IL-12 mAb. RNA was analyzed byPCR for TIM gene expression.

TIM-3 was generally expressed after Th1 differentiation whereas TIM-1was lost. Conversely, TIM-3 was not expressed in any of the Th2 butTIM-1 was expressed in all Th2 cells. Both TIM-1 and TIM-3 are expressedin monocyte-depleted, unstimulated peripheral blood mononuclear cellsfrom the donors used to derive the Th1 and Th2 cell lines, presumablybecause this mixed population contains both Th1 and Th2 memory cells.These results suggest a reciprocal relationship with TIM-1 beingexpressed in Th2 and TIM-3 in Th1. This reciprocal relationship betweenTIM-1 and TIM-3 has also been observed in the mouse.

In human tissues, a 4.4 kb TIM-1 mRNA was very strongly expressed inkidney and testis. The 4.4-kb mRNA was present in almost all tissues,though it was faint in most. A 5.5-kb band was observed in colon andliver. A 7.5-kb band was observed in spleen, thymus, and peripheralblood leukocytes, and smaller than 4.4-kb bands were observed in someorgans. These results suggest that hTIM-1 is expressed at some level inmost human tissues and that a message of 7.5-kb may code for hTIM-1 intissues of immunological interest. However, expression of Kim-1 (KidneyInjury Molecule-1), the rat homologue of TIM-1, increases in kidney uponischemic injury. Since the MTN blots used in the expression analysiswere prepared from mRNA extracted from cadavers, the increasedexpression of TIM-1 in kidney was re-analyzed. TIM-1 was not found to beoverexpressed in kidney RNA obtained from normal kidney biopsies.Therefore, it is likely that the high levels of expression of TIM-1observed in kidney and testis were due to an up-regulation in theexpression of TIM-1 resulting from tissue injury. The injured kidney mayexpress proteins that direct incoming inflammatory cells towards a moreprotective Th2 response rather than a destructive Th1 response.

Example 4 TIM Ligands and Antibodies

Generation of Antibodies. Generation of monoclonal antibodies againstmouse TIM-1 allows examination of the cell surface expression of TIM-1in different tissues, cell lines and mouse strains. Both alleles ofmouse TIM-1 have been cloned into a vector for high protein expression(Invitrogen, pEF6-TOPO). Rats have been immunized and boosted with bothTim1 cDNA constructs to rapidly generate antibodies against cell surfacemolecules. This method with cDNA vaccination favors the production ofmAb against cell surface epitopes since the Tim1 cDNA will be taken upby APC, which will express the TIM-1 as a cell surface molecule. Inorder to generate mAb that would bind equally well to both the BALB/cand the HBA TIM-1 (by binding to conserved domains of TIM-1 such as theImmunoglobulin domain of TIM-1), both the BALB/c and HBA Tim1 cDNA(pEF6-mTIMbalb and pEF6-mTIMhba) were injected into each rat.

Further boosting of the Tim1 cDNA-immunized rats was done with CHO cellsstably transfected with the pEF6-mTIM-1-GFP expression constructs. CHOtransfectants expressing high levels of mouse TIM-1 were sorted by FACS,and injected into the rats. Another mTIM-1 expressing cell was generatedby stably transfecting the pre-B cell line 300.19 with the pEF6-mTIM-1expression constructs. This line is used to screen the rat serum and thehybridomas following fusion for anti-TIM-1 antibody by flow cytometry.Rats have been generated which have high polyclonal titers againstanti-TIM-1, as detected by the binding of rat serum (and a secondaryFITC-goat anti-rat Ig) to stable pEF6-mTIM1-transfected 300.19 cells, ascompared with control serum from unimmunized rats. This staining isspecific for TIM-1 since there is no reactivity with nontransfectedcells or cells transfected with TIM-2.

The rat spleen is removed and the splenocytes fused with a myeloma cellline (SP/2) to produce hybridomas. Hybridoma supernatants are screenedusing the TIM-1 transfected 300.19 cell lines to identify hybridomaclones that produce monoclonal anti-TIM-1. Specificity of the mAb forTIM-1 (and not other TIM proteins) is confirmed using TIM-2 transfectedcells and mTIM-3 transfected cells or TIM-31 g fusion protein.

Antibody Staining. Th1 and Th2 cell lines were generated from bothBALB/c and HBA DO11.10 spleen cells. RT-PCR for TIM-1 mRNA expressiondemonstrated that TIM-1 is expressed in Th2 lines, but not in Th1 lines,following two rounds of restimulation with antigen under standardpolarizing conditions. DO11.10 T cells following two rounds ofstimulation with antigen/APC under Th2 polarizing conditions werestained with the polyclonal rat anti-TIM-1 antiserum. These Th2 cellsexpressed high levels of TIM-1.

These experiments showing preferential expression of TIM-1 in Th2 linesare quantified and confirmed using anti-Tim-1 mAbs and Northern blots.DO11.10 cells from BALB and HBA are cultured with antigen and APC, andrestimulated for 1, 2, and 3 weeks under standard polarizing conditions(anti-IL-12 plus IL-4 or anti-IL-4 plus IL-12). After each week ofstimulation, cells are stained with anti-TIM-1 mAb. By harvestingstimulated cells at various time points the kinetics of TIM-1 expressionon T cells undergoing differentiation to Th1 or Th2 subset isdetermined. To determine if Tim-1 surface expression changes following Tcell activation, we will also compare TIM-1 expression on resting andactivated T cells one week after each round of antigen stimulation, bystimulating some cells with PMA and ionomycin. Activated cells arestained for intracellular cytokine expression to verify the Th subsetdifferentiation of the T cells. Alternatively, quantitative RT-PCR ornorthern blots using mRNA harvested from T cells activated with PMA plusionomycin, following each round of stimulation, are used to determinerelative levels of mRNA production.

TIM-1-Ig fusion proteins BALB/c TIM-1-mIgG2a has been prepared, which isa fusion protein between the TIM-1 polypeptide and the Fc region ofmouse immunoglobulin. The vector has been engineered to contain amutation in murine IgG2a Fc that minimizes binding to Fc receptors. TheTIM-1 fusion protein is utilized in characterization of TIM-1 function.The TIM-1 Ig fusion protein is expected to block TIM-1 function bybinding to the TIM-1 ligand and interrupt TIM-1/TIM-1-ligandinteractions.

Purified D1muc-Fc fusion protein containing the cys-rich immunoglobulindomain and ⅔ of the mucin-like region of TIM-1 fused to the hinge and Fcfragment of human IgG1 (IgVmuc-hIg) was run on a gel. This protein wasexpressed in CHO cells, and the IgVmuc-hIg protein was purified from CHOsupernatants with protein-A agarose columns. Purified IgVmuc-hIg fusionprotein neutralizes about 2 logs of HAV infectivity. In addition,treatment of HAV with IgVmuc-hIg produced a major shift in thesedimentation of the HAV particles, indicating that IgVmuc-hIg induceduncoating of the viral genome, whereas a fusion protein containing onlythe Ig-like region without the mucin domain (IgV-hIg) did not. This HAVneutralization system and the IgVmuc-hIg fusion protein will be used toanalyze the function of TIM-1/HAV receptor alleles.

Based upon the in vivo effect of anti-TIM-3 mAb on macrophage expansionand activation, it was hypothesized that the TIM-3 ligand would beexpressed on cells of the myeloid lineage. Dendritic cells (DC) wereprepared from blood monocytes according to established protocols with1000 U/ml IL-4 and 800 U/ml GM-CSF. DC were matured by replating thecells for 2 days in IL-4 (1000 U/ml) and GM-CSF (800 U/ml) supplementedwith IL-1β (10 ng/ml), TNF-α (10 ng/ml), IL-6 (1000 U/ml), and PGE₂ (1μg/ml). Mature DCs stained positively with hTIM-3-Ig, though there wasvariability among donors, suggesting that mature DC express a ligand forthe IgV domain of TIM-3. Bone marrow derived endothelial cells stainedvery weakly and B cell lines did not stain with TIM-3-Ig.

Although the intracytoplasmic tail of Tim1/huhavcr-1 is relativelyshort, it contains a sequence that is highly conserved between mouse,rat, human and monkey (SEQ ID NO:59, RAEDNIYI), and which may bephosphorylated, and signal through interaction with other signaltransduction molecules. The most likely candidate molecule that can bindthe RAEDNIYI motif in T cells is the tyrosine kinase Itk. Theinterleukin-2 inducible tyrosine kinase, Itk is a nonreceptor proteintyrosine kinase of the Tec family that participates in the intracellularsignaling events leading to T cell activation. Tec family memberscontain the conserved SH3, SH2, and catalytic domains common to manykinase families, but they are distinguished by unique sequences outsideof this region. It is known that Itk phosphorylate phospholipase C-γ(PLC-γ), and triggers a cascade of signaling events that are involved inT cell activation and helper T cell differentiation. In the absence ofItk signaling, Th2 cells do not develop. These results suggest thatTIM-1/huhavcr-1 may signal through Itk, thereby altering the cytokinedevelopment in CD4 T cells.

Example 5 Combination of TIM Activation with Cytotherapy

The induction of liver cell death in a mouse model of hepatitis orairway cell death in a model of airway reactivity promotes TIM-1mediated costimulation by PS (Lee et al. (2010) J. Immunol.185(9):5225-35). As co-ligands for phosphatidylserine (PS), TIM-3 andTIM-4 (which is a counter ligand for TIM-1) further modulate immuneresponses in these settings via PS-binding. TIM-directed therapies canmodify responses to cytotoxic treatments, including chemotherapy orradiation (such as radiation-induced pneumonitis, hepatitis, and otherradiation-specific effects) and modify anti-tumor immune responses,especially after radiation. The identity of PS as a ligand for the TIMfamily members creates opportunities for imaging TIM-binding capacity invivo and adapting therapies accordingly.

As described in Example 4, antibodies have been produced against mouseTIM-1. Additionally antibodies have been produced that specifically bindto human TIM-1 (Umetsu et al (2005) Nature Immunology 6, 447-454),providing a library of well characterized monoclonal antibodies againsthuman and mouse TIM-1. The agonistic monoclonal 3B3 antibody blocksTIM-1:PS binding and costimulates T and NKT cells as an immune therapy.When used as tumor immunotherapy (IT) and when combined with radiation(RT) to the primary tumor site, this antibody enhances local breastcancer tumor control and reduces the size and number of distant breastcancer metastases in a mouse model of breast cancer. The combinationtherapy may improve outcomes for patients who receive radiation formetastatic or locally advanced breast cancer by inducing immuneresponses against their tumors.

Because TIM-1 is activated by binding to phosphatidylserine, aphospholipid that is exposed in irradiated tumor tissues, IT with TIM-1may demonstrate greater specificity (and fewer ‘off target’ autoimmuneside effects) than other IT agents currently under development, and thefunctional interaction between TIM-1 and PS in the tumor immunemicroenvironment provides a unique method for focusing immune activationto the irradiated tumor.

The use of imaging tracers coupled to PS-binding molecules, such asannexin V or TIM-containing peptides or mimics, can be used to monitorand optimize the delivery of immunotherapies that target the PS-bindingTIM family members.

Example 5 Tumor Model Studies

Our studies identified specific, well defined changes in thesubpopulations of cells present after radiation therapy (RT) and thecostimulatory receptors expressed those cells. We find that RTselectively induces the expression of certain receptors, such as theTIMs, CD40, 4-1 BB, PD-1, and PD-L1. This enhancement persisted for atleast one week, which includes the 24-72 hour timeframe that is criticalfor initiation of anti-tumor immune responses after RT. Our resultsindicated that these targets (PD1/PD-L1 and TIM-1) could be moreeffective in combination with RT than targets such as CTLA-4 that arenot as significantly upregulated after RT. The relative radioresistanceof regulatory T cell (Treg), myeloid suppressor cell (MSC), and antigenpresenting cell (APC) subsets, also made them attractive targets forimmunotherapy after RT. Thus, studies were performed with theexperimental design shown in FIG. 9.

The most efficacious regimens identified in these studies targetedTIM-1. In mice with established, metastatic breast cancer,administration of an activating anti-TIM1 antibody at the time ofprimary tumor irradiation resulted in decreased metastatic diseaseburden and increased overall survival, compared to mice treated with RTalone, while the antibody alone had no effect, shown in FIG. 10.Specifically, administration of an activating anti-TIM1 (α-TIM1, 3B3)antibody at the time of primary tumor RT leads to: 1) decreasedmetastatic disease burden (shown in FIG. 11 with day 55 liver histology;and in FIGS. 12) and 2) increased overall. This RT+IT combinationproduces a robust anti-breast cancer vaccination effect, leading toeradication of metastases outside of the radiation field. Importantly,this benefit was observed without systemic or local (in the irradiatedfield) tissue toxicities. Inhibition of PD-L1 and depletion of MSCs didnot demonstrate a therapeutic effect in this model.

These data show that RT has substantial effects on immune cells that arecritical for IT, and identify TIM1 as an especially effective IT tocombine with RT. These changes provide a window for the systematicmanipulation of anti-breast cancer immune responses. TIM-1 is anespecially attractive IT target for use with RT for the treatment ofmetastatic or locally advanced, unresectable breast cancers.

Example 6 Effects of Irradiation and Hematopoetic Stem CellTransplantation on Airway Hyperreactivity

Airway hyperreactivity (AHR) is a complex trait, influenced by multiplegenetic and environmental factors, which are the subject of active basicand clinical research. Diffuse pulmonary toxicity is a seriouscomplication of myeloablative hematopoetic stem cell transplantation(HCT) regimens, but our understanding of the etiology of this is verylimited. Few case reports document transfer of atopic disorders afterallogeneic HCT, and evidence for this in animal models is limited. Inpediatric patients undergoing myeloablative autologous HCT for Hodgkinsdisease, 44% of patients develop diffuse pulmonary toxicity. Thiscomplication was not related to the method of transplantationconditioning (irradiation versus cytotoxic chemotherapy), but wasassociated with a pretransplantation history of atopy (allergic rhinitisor asthma), with an 80% incidence among those with an atopic historycompared with 20% among those without an atopic history. Whileinvestigating the genetic causes of AHR in mice, we discovered twopreviously unidentified determinants of AHR following myeloablative andnonmyeloablative transplantation conditioning protocols usingirradiation.

Materials/Methods:

BALB/c (atopic) and BALB/c congenic C.D2Es-Hba (HBA) mice (non-atopic)were irradiated with nonmyeloablative (450-550 Rad) or myeloablative(900 Rad) doses prior to adoptive transfer of splenocytes (SPC) or bonemarrow cells (BMC), respectively. Three days following the celltransfer, a standard airway sensitization protocol was initiated, usingovalbumin and alum i.p., followed one week later by three daily doses ofintranasal ovalbumin (50 ug/ml). Airway hyperreactivity (Penh) wasassessed by whole body plethysmography with increasing challenges ofnebulized methacholine. After airway testing, the mice were euthanizedand lung tissues were obtained for histology.

Results: Airway hyperreactivity was induced in BALB/c mice reconstitutedwith BALB/c SPC or BMC. However, non-atopic HBA mice resisted thedevelopment of AHR, whether reconstituted with BALB/c or HBA SPC or BMC.BALB/c reconstituted with HBA cells resisted AHR with Penh levelscomparable to HBA mice reconstituted with HBA cells. We were surprisedto find that the timing of cell transfer and immunization influenced thedevelopment of airway hyperreactivity. Cell transfers performed within1-2 hours of irradiation were associated with the development of AHR inall mice, while transfer between 8-12 hours after irradiation did notmodify the airway sensitization.

CONCLUSIONS

Pulmonary toxicity following HCT is a complex phenomenon, influenced bygenetic factors and by transplantation protocol. Our results in miceconfirm retrospective observations in humans that atopic predispositionis a major risk factor for the development of airway hyperreactivityafter transplantation. These studies are the first to demonstrate thatone of the multiple factors involved in susceptibility to pulmonarytoxicity after transplantation is a radioresistant cell population thatcan be adoptively transferred with spleen or bone marrow cells. We alsodemonstrate that the timing of HCT cell transfusion contributes to thedevelopment of airway hyperreactivity.

What is claimed is:
 1. A method for the treatment of malignancy in anindividual, the method comprising: administering to said individual anagonistic antibody or fragment thereof that specifically binds to ahuman T cell/transmembrane, immunoglobulin, and mucin (TIM) encodedpolypeptide selected from TIM-1 polypeptide and human TIM-4 polypeptide,in combination with radiation therapy.
 2. The method according to claim1, wherein said TIM polypeptide is TIM-1.
 3. The method according toclaim 1, wherein said TIM polypeptide is TIM-4.
 4. The method of claim1, wherein distribution of phosphatidylserine (PS) in the individual isdetermined prior to or in conjunction with administering said antibodyor fragment thereof, wherein an individual in which the PS is bound to atumor cell of the malignancy is selected for administration of theantibody or fragment thereof.
 5. The method of claim 4, wherein thedistribution of phosphatidylserine is determined by in vivo imaging witha labeled phosphatidylserine binding agent.
 6. A method for thetreatment of malignancy in an individual, the method comprising:administering to said individual an antagonistic antibody or fragmentthereof that specifically binds to human T cell/transmembrane,immunoglobulin, and mucin (TIM) encoded polypeptide TIM-3 in combinationwith radiation therapy to a local irradiation field, wherein theantagonistic antibody is provided at a dose that boosts immuneresponsiveness after radiation therapy to generate a response to sitesof metastatic disease outside of the irradiation field.